Methods and compositions for treating huntington&#39;s disease

ABSTRACT

Disclosed herein are compositions comprising non-naturally occurring zinc finger domains, fusion proteins comprising these zinc finger domains, polynucleotides encoding these proteins, cells expressing these proteins and pharmaceutical compositions comprising these proteins or polynucleotides as well as methods of modifying an Htt gene using these compositions for treating or preventing Huntington&#39;s Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/294,062, filed Oct. 14, 2016, which is a continuation ofU.S. patent application Ser. No. 14/446,189, filed Jul. 29, 2014, nowU.S. Pat. No. 9,499,597 which is a continuation of U.S. patentapplication Ser. No. 13/781,176, filed Feb. 28, 2013, now U.S. Pat. No.8,841,260, which claims the benefit of U.S. Provisional PatentApplication No. 61/605,028, filed Feb. 29, 2012, the disclosures ofwhich are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present disclosure is in the fields of gene expression and genomeediting.

BACKGROUND

Huntington's Disease (HD), also known as Huntington's Chorea, is aprogressive disorder of motor, cognitive and psychiatric disturbances.The mean age of onset for this disease is age 35-44 years, although inabout 10% of cases, onset occurs prior to age 21, and the averagelifespan post-diagnosis of the disease is 15-18 years. Prevalence isabout 3 to 7 among 100,000 people of western European descent.

Huntington's Disease is an example of a trinucleotide repeat expansiondisorders were first characterized in the early 1990s (see Di Prosperoand Fischbeck (2005) Nature Reviews Genetics 6:756-765). These disordersinvolve the localized expansion of unstable repeats of sets of threenucleotides and can result in loss of function of the gene in which theexpanded repeat resides, a gain of toxic function, or both.Trinucleotide repeats can be located in any part of the gene, includingnon-coding and coding gene regions. Repeats located within the codingregions typically involve either a repeated glutamine encoding triplet(CAG) or an alanine encoding triplet (CGA). Expanded repeat regionswithin non-coding sequences can lead to aberrant expression of the genewhile expanded repeats within coding regions (also known as codonreiteration disorders) may cause mis-folding and protein aggregation.The exact cause of the pathophysiology associated with the aberrantproteins is often not known. Typically, in the wild-type genes that aresubject to trinucleotide expansion, these regions contain a variablenumber of repeat sequences in the normal population, but in theafflicted populations, the number of repeats can increase from adoubling to a log order increase in the number of repeats. In HD,repeats are inserted within the N terminal coding region of the largecytosolic protein Huntingtin (Htt). Normal Htt alleles contain 15-20 CAGrepeats (SEQ ID NO: 226), while alleles containing 35 or more repeats(SEQ ID NO: 227) can be considered potentially HD causing alleles andconfer risk for developing the disease. Alleles containing 36-39 repeats(SEQ ID NO: 228) are considered incompletely penetrant, and thoseindividuals harboring those alleles may or may not develop the disease(or may develop symptoms later in life) while alleles containing 40repeats or more (SEQ ID NO: 259) are considered completely penetrant. Infact, no asymptomatic persons containing HD alleles with this manyrepeats have been reported. Those individuals with juvenile onset HD(<21 years of age) are often found to have 60 or more CAG repeats (SEQID NO: 229). In addition to an increase in CAG repeats, it has also beenshown that HD can involve +1 and +2 frameshifts within the repeatsequences such that the region will encode a poly-serine polypeptide(encoded by AGC repeats in the case of a +1 frameshift) track ratherthan poly-glutamine (Davies and Rubinsztein (2006) Journal of MedicalGenetics 43:893-896).

In HD, the mutant Htt allele is usually inherited from one parent as adominant trait. Any child born of a HD patient has a 50% chance ofdeveloping the disease if the other parent was not afflicted with thedisorder. In some cases, a parent may have an intermediate HD allele andbe asymptomatic while, due to repeat expansion, the child manifests thedisease. In addition, the HD allele can also display a phenomenon knownas anticipation wherein increasing severity or decreasing age of onsetis observed over several generations due to the unstable nature of therepeat region during spermatogenesis.

Furthermore, trinucleotide expansion in Htt leads to neuronal loss inthe medium spiny gamma-aminobutyric acid (GABA) projection neurons inthe striatum, with neuronal loss also occurring in the neocortex. Mediumspiny neurons that contain enkephalin and that project to the externalglobus pallidum are more involved than neurons that contain substance Pand project to the internal globus pallidum. Other brain areas greatlyaffected in people with Huntington's disease include the substantianigra, cortical layers 3, 5, and 6, the CA1 region of the hippocampus,the angular gyms in the parietal lobe, Purkinje cells of the cerebellum,lateral tuberal nuclei of the hypothalamus, and thecentromedialparafascicular complex of the thalamus (Walker (2007) Lancet369:218-228).

The role of the normal Htt protein is poorly understood, but it may beinvolved in neurogenesis, apoptotic cell death, and vesicle trafficking.In addition, there is evidence that wild-type Htt stimulates theproduction of brain-derived neurotrophic factor (BDNF), a pro-survivalfactor for the striatal neurons. It has been shown that progression ofHD correlates with a decrease in BDNF expression in mouse models of HD(Zuccato, et al. (2005) Pharmacological Research 52(2):133-139), andthat delivery of either BDNF or glial cell line-derived neurotrophicfactor (GDNF) via adeno-associated viral (AAV) vector-mediated genedelivery may protect straital neurons in mouse models of HD (Kells, etal. (2004) Molecular Therapy 9(5):682-688).

Treatment options for HD are currently very limited. Some potentialmethodologies designed to prevent the toxicities associated with proteinaggregation that occurs through the extended poly-glutamine tract suchas overexpression of chaperonins or induction of the heat shock responsewith the compound geldanamycin have shown a reduction in thesetoxicities in in vitro models. Other treatments target the role ofapoptosis in the clinical manifestations of the disease. For example,slowing of disease symptoms has been shown via blockage of caspaseactivity in animal models in the offspring of a pairing of mice whereone parent contained a HD allele and the other parent had a dominantnegative allele for caspase 1. Additionally, cleavage of mutant HD Httby caspase may play a role in the pathogenicity of the disease.Transgenic mice carrying caspase-6 resistant mutant Htt were found tomaintain normal neuronal function and did not develop striatalneurodegeneration as compared to mice carrying a non-caspase resistantmutant Htt allele (see Graham, et al. (2006) Cell 125:1179-1191).Molecules which target members of the apoptotic pathway have also beenshown to have a slowing affect on symptomology. For example, thecompounds zVAD-fmk and minocycline, both of which inhibit caspaseactivity, have been shown to slow disease manifestation in mice. Thedrug remacemide has also been used in small HD human trials because thecompound was thought to prevent the binding of the mutant Htt to theNDMA receptor to prevent the exertion of toxic effects on the nervecell. However, no statistically significant improvements were observedin neuron function in these trials. In addition, the Huntington StudyGroup conducted a randomized, double-blind study using Co-enzyme Q.Although a trend towards slower disease progression among patients thatwere treated with coenzyme Q10 was observed, there was no significantchange in the rate of decline of total functional capacity. (Di Prosperoand Fischbeck, ibid). U.S. Patent Publication No. 2011/0082093 disclosesspecific zinc finger proteins targeted to Htt.

Thus, there remains a need for compositions and methods for thetreatment and prevention of Huntington's Disease.

SUMMARY

Disclosed herein are methods and compositions for treating Huntington'sDisease. In particular, provided herein are methods and compositions formodifying (e.g., modulating expression of) an HD Htt allele so as totreat Huntington Disease. Also provided are methods and compositions forgenerating animal models of Huntington's Disease.

Thus, in one aspect, engineered DNA binding domains (e.g., zinc fingerproteins or TAL effector (TALE) proteins) that modulate expression of aHD allele (e.g., Htt) are provided. Engineered zinc finger proteins orTALEs are non-naturally occurring zinc finger or TALE proteins whose DNAbinding domains (e.g., recognition helices or RVDs) have been altered(e.g., by selection and/or rational design) to bind to a pre-selectedtarget site. Any of the zinc finger proteins described herein mayinclude 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in the selectedsequence(s) (e.g., gene(s)). Similarly, any of the TALE proteinsdescribed herein may include any number of TALE RVDs. In someembodiments, at least one RVD has non-specific DNA binding. In someembodiments, at least one recognition helix (or RVD) is non-naturallyoccurring. In certain embodiments, the zinc finger proteins have therecognition helices shown in Tables 1A and 1B. In other embodiments, thezinc finger proteins bind to the target sequences shown in Tables 2A and2B. In some embodiments, the zinc finger proteins comprise therecognition helices in Table 2C. In certain embodiments, the zinc fingerproteins are formulated into a pharmaceutical composition, for example,for administration to a subject.

In one aspect, repressors (ZFP-TFs or TALE-TFs) are provided that bindto sequences entirely or partially outside the CAG repeat region of Htt.In another aspect, ZFP or TALE repressors (ZFP-TFs or TALE-TFs) areprovided that bind to sequences within CAG repeat region of Htt. In someembodiments, these ZFP-TFs or TALE-TFs preferentially bind to expandedtrinucleotide tracts relative to repeat tracts of a wild-type length,thereby achieving preferential repression of the expanded allele. Insome embodiments these ZFP-TFs or TALE-TFs include protein interactiondomains (or “dimerization domains”) that allow multimerization whenbound to DNA. In some embodiments, these ZFP-TFs or TALE TFs achievecooperative DNA binding to the repeat sequence so that the expandedallele is bound more efficiently by a larger number of ZFPs or TALEproteins than the wild-type allele, allowing preferential repression ofthe mutant allele. These cooperative binding ZFP-TFs or TALE TFs may ormay not further contain protein interaction domains that allowmultimerization when bound to DNA. In some embodiments, ZFP TFs or TALETFs form a stable complex of multimers of a given size, and thus arecapable of preferentially interacting with a CAG tract above a certainminimum size, wherein that minimum size is greater than the length of awild-type CAG tract.

In certain embodiments, the ZFPs or TALE proteins as described herein(e.g., two-handed, multimerizing, etc.) preferentially modify expressionof a mutant Htt allele. In some embodiments, the ZFP or TALE bindsspecifically to mutant Htt alleles wherein the expanded tract encodespoly-glutamine, while in other embodiments, the ZFP or TALE bindsspecifically to a mutant Htt allele wherein the expansion tract encodespoly-serine. Thus, in some embodiments, the ZFP-TF or TALE-TF modulatesboth the wild type and mutant forms of the Htt allele. In certainembodiments, the ZFP or TALE modulates only the wild type Htt allele. Inother embodiments, the ZFP or TALE modulates only the mutant form ofHtt.

In other embodiments, repressing ZFP-TFs or TALE-TFs are provided whichpreferentially bind to known SNPs associated with the expanded HD Httalleles. In this way, the ZFP-TFs or TALE-TFs are specific for mutantHtt alleles which contain the SNP, allowing for specific repression ofthe mutant Htt allele. In another aspect, ZFP-TFs or TALE-TFs thatspecifically activate the wild-type Htt allele by interacting with SNPsassociated with wild-type alleles are provided. In this way, only thewild-type Htt allele is activated.

In certain embodiments, the zinc finger proteins (ZFPs) or TALE proteinsas described herein can be placed in operative linkage with a regulatorydomain (or functional domain) as part of a fusion protein. Thefunctional domain can be, for example, a transcriptional activationdomain, a transcriptional repression domain and/or a nuclease (cleavage)domain. By selecting either an activation domain or repression domainfor fusion with the ZFP or TALE, such fusion proteins can be used eitherto activate or to repress gene expression. In some embodiments, a fusionprotein comprising a ZFP or TALE targeted to a mutant Htt as describedherein fused to a transcriptional repression domain that can be used todown-regulate mutant Htt expression is provided. In some embodiments, afusion protein comprising a ZFP or TALE targeted to a wild-type Httallele fused to a transcription activation domain that can up-regulatethe wild type Htt allele is provided. In certain embodiments, theactivity of the regulatory domain is regulated by an exogenous smallmolecule or ligand such that interaction with the cell's transcriptionmachinery will not take place in the absence of the exogenous ligand.Such external ligands control the degree of interaction of the ZFP-TF orTALE-TF with the transcription machinery. The regulatory domain(s) maybe operatively linked to any portion(s) of one or more of the ZFPs orTALEs, including between one or more ZFPs or TALEs, exterior to one ormore ZFPs or TALEs and any combination thereof. Any of the fusionproteins described herein may be formulated into a pharmaceuticalcomposition.

In some embodiments, the engineered DNA binding domains as describedherein can be placed in operative linkage with nuclease (cleavage)domains as part of a fusion protein. In other embodiments, nucleasesystems such as the CRISPR/Cas system may be utilized with a specificsingle guide RNA to target the nuclease to a target location in the DNA.In certain embodiments, such nucleases and nuclease fusions may beutilized for targeting mutant Htt alleles in stem cells such as inducedpluripotent stem cells (iPSC), human embryonic stem cells (hESC),mesenchymal stem cells (MSC) or neuronal stem cells wherein the activityof the nuclease fusion will result in an Htt allele containing a wildtype number of CAG repeats. In certain embodiments, pharmaceuticalcompositions comprising the modified stem cells are provided.

In yet another aspect, a polynucleotide encoding any of the DNA bindingproteins described herein is provided. In another aspect,polynucleotides encoding a CRIPSR/Cas nuclease and a single guide RHAare provided. Such polynucleotides can be administered to a subject inwhich it is desirable to treat Huntington's Disease.

In still further aspects, the invention provides methods andcompositions for the generation of specific model systems for the studyof Huntington's Disease. In certain embodiments, provided herein aremodels in which mutant Htt alleles are generated using embryonic stemcells to generate cell and animal lines in which trinucleotide expansiontracts of specific lengths (50 (SEQ ID NO: 230), 80 (SEQ ID NO: 231),109 (SEQ ID NO: 232) and 180 (SEQ ID NO: 233) CAG repeats, for example)are inserted into a wild-type Htt allele using zinc finger nuclease(ZFN), TALE-nuclease (TALEN), or CRISPR/Cas nuclease driven targetedintegration. In certain embodiments, the model systems comprise in vitrocell lines, while in other embodiments, the model systems comprisetransgenic animals. In any of the animal models described herein, theanimal may be, for example, a rodent (e.g., rat, mouse), a primate(e.g., non-human primate) or a rabbit.

In yet another aspect, a gene delivery vector comprising any of thepolynucleotides described herein is provided. In certain embodiments,the vector is an adenovirus vector (e.g., an Ad5/F35 vector), alentiviral vector (LV) including integration competent orintegration-defective lentiviral vectors, or an adenovirus associatedviral vector (AAV). Thus, also provided herein are adenovirus (Ad)vectors, LV or adenovirus associate viral vectors (AAV) comprising asequence encoding at least one nuclease (ZFN or TALEN) and/or a donorsequence for targeted integration into a target gene. In certainembodiments, the Ad vector is a chimeric Ad vector, for example anAd5/F35 vector. In certain embodiments, the lentiviral vector is anintegrase-defective lentiviral vector (IDLV) or an integration competentlentiviral vector. In certain embodiments the vector is pseudo-typedwith a VSV-G envelope, or with other envelopes.

In some embodiments, model systems are provided for Huntington's diseasewherein the target alleles (e.g., mutant Htt) are tagged with expressionmarkers. In certain embodiments, the mutant alleles (e.g., mutant Htt)are tagged. In some embodiments, the wild type allele (e.g., wild-typeHtt) is tagged, and in additional embodiments, both wild type and mutantalleles are tagged with separate expression markers. In certainembodiments, the model systems comprise in vitro cell lines, while inother embodiments, the model systems comprise transgenic animals.

Additionally, pharmaceutical compositions comprising the nucleic acidsand/or proteins (e.g., ZFPs or TALEs or fusion proteins comprising theZFPs or TALEs) are also provided. For example, certain compositionsinclude a nucleic acid comprising a sequence that encodes one of theZFPs or TALEs described herein operably linked to a regulatory sequence,combined with a pharmaceutically acceptable carrier or diluent, whereinthe regulatory sequence allows for expression of the nucleic acid in acell. In certain embodiments, the ZFPs or TALEs encoded are specific fora HD Htt allele. In some embodiments, pharmaceutical compositionscomprise ZFPs or TALEs that modulate a HD Htt allele and ZFPs or TALEsthat modulate a neurotrophic factor. Protein based compositions includeone of more ZFPs or TALEs as disclosed herein and a pharmaceuticallyacceptable carrier or diluent.

In yet another aspect also provided is an isolated cell comprising anyof the proteins, polynucleotides and/or compositions as describedherein.

In another aspect, provided herein are methods for treating and/orpreventing Huntington's Disease using the methods and compositionsdescribed herein. In some embodiments, the methods involve compositionswhere the polynucleotides and/or proteins may be delivered using a viralvector, a non-viral vector (e.g., plasmid) and/or combinations thereof.In some embodiments, the methods involve compositions comprising stemcell populations comprising a ZFP or TALE, or altered with the ZFNs,TALENs or the CRISPR/Cas nuclease system of the invention.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1E are schematics depicting wild type and mutant(Huntington's Disease, HD) Huntingtin (Htt) allele and various ZFP-TFsbinding to those alleles. FIG. 1A shows ZFP designs that bind outside ofthe CAG repeat, and therefore are predicted to bind equally to the wildtype allele and the mutant (HD) allele. “KRAB” refers to the KRABrepression domain from the KOX1 gene and “ZFP” refers to the zinc fingerDNA binding protein. “Standard ZFP TF” is a ZFP transcription factorfusion protein in which the zinc finger DNA binding domains are linkedto the KRAB repression domain. FIG. 1B shows ZFP-TFs designed to bindwithin the CAG region. FIG. 1C depicts a “two-handed ZFP TF,” which is aZFP transcription factor in which two clusters of zinc finger domainsare separated by a rigid protein sequences. The functional (repression)domain is depicted exterior to one ZFP in this Figure, but it will beapparent that the functional domain may be between the ZFPs or exteriorto the ZFPs on either end of the protein. FIG. 1D depicts a“multimerizing ZFP TF,” which is a ZFP TF that is capable ofmultimerizing through a multimerization domain (depicted as speckledboxes). FIG. 1E depicts a ZFP-ZFP-KRAB configuration where two zincfinger DNA binding domains are linked by a flexible linker and are alsofused to a KRAB domain. It will be apparent to the skilled artisan thatin all fusion proteins, the functional domain can be on either end ofthe DNA binding domain, and that the DNA binding domain may comprise awide number range of zinc fingers. Also depicted in FIG. 1 as a box withblack diamonds is a functional domain (e.g., activation, repression,cleavage domain). It will be apparent to the skilled artisan that theexemplary models presented in the Figures may apply to TALE TFs as well.

FIGS. 2A through 2E depict the repression of both alleles of Htt by ZFPsas described in FIG. 1A using ZFP TFs that do not bind to the CAG repeatsequences. The ZFP identification numbers as shown in Tables 1A and 1Bare indicated below the bars. FIG. 2A depicts repression of the humanHtt alleles in HEK293 cells using ZFPs targeted to five loci in thehuman gene. A diagram of the human Htt gene is shown and the locationsof ZFP binding sites are shown. For each ZFP group, each bar representsan independent transfection. FIG. 2B depicts a Western blot showing Httprotein levels in HEK293 cells transfected with the GFP control or the18856 ZFP TF repressor (comprising the KRAB repression domain of KOX1),where the NFκB p65 levels (“p65”) were used to confirm equal proteinloading. The Western blot confirms the repression of Htt expression bythe ZFP-TF. FIG. 2C depicts a similar set of data as FIG. 2A for themouse Htt specific ZFP in Neuro2A cells. As in FIG. 2A, a diagram of themouse Htt gene is shown and the locations of ZFP binding sites areindicated. FIGS. 2D and 2E demonstrate the repression of mouse Htt geneexpression (RNA) in immortalized striatal cells, where different dosesof ZFP-TF mRNA were used for transfection. In all cases except FIG. 2B,Htt mRNA levels were measured by real-time RT-PCR and normalized tothose of Actin mRNA.

FIGS. 3A through 3G depict selective repression of mutant Htt by usingZFPs binding within the CAG repeat region, as illustrated in FIG. 1B.This model illustrates that the longer CAG repeat region in the mutantallele allows for increased binding of CAG-targeted ZFP repressormolecules. FIG. 3A depicts different repressor activities on theendogenous Htt gene (with normal CAG repeat length) by CAG-targeted ZFPsin HEK293 cells. FIG. 3B shows repression of luciferase reporterscontrolled by Htt promoter/exon1 fragments containing CAG repeats ofvarying lengths, ranging from 10 (SEQ ID NO: 234) to 47 (SEQ ID NO: 235)CAG repeats. CAG10 (SEQ ID NO: 234) (left-most bar for each of the twoindicated conditions) shows results with 10 CAG repeats (SEQ ID NO:234); CAG17 (SEQ ID NO: 236) (bar second from the left for each of thetwo indicated conditions) shows results with 17 CAG repeats (SEQ ID NO:236); CAG23 (SEQ ID NO: 237) (bar second from the right for each of thetwo indicated conditions) shows results with 23 CAG repeats (SEQ ID NO:237); and CAG47 (SEQ ID NO: 235) (right-most bar for each of the twoindicated conditions) shows results with 47 CAG repeats (SEQ ID NO:235). The schematic above the graph depicts the arrangement of the Httpromoter, exon 1, the CAG repeats and the reporter luciferase gene usedin this system. The data demonstrate that increasing the number of CAGsleads to a decreased expression from the Htt promoter by a CAG-targetedZFP. Furthermore, FIG. 3C demonstrates that, while a relatively weakCAG-targeted ZFP does not repress the luciferase reporter that containsa normal-length CAG repeat as well as a strong CAG repressor, it drivessimilar repression of a luciferase reporter that contains an expandedCAG repeat as the strong CAG-targeted ZFP at all doses tested.“pRL-Htt-CAG23-intron 1” (“CAG23” disclosed as SEQ ID NO: 237) (left barof each pair) corresponds to expression from the wild type allele while“pRL-HttCAG47-intron 1” (“CAG47” disclosed as SEQ ID NO: 235) (right barof each pair) correlates with expression from the mutant expanded Httallele (containing 47 CAG repeats (SEQ ID NO: 235)). FIG. 3D is a graphdepicting repression of mutant Htt (111 CAG (SEQ ID NO: 238)) byCAG-targeted ZFPs in immortalized mouse striatal cells derived fromHdH(Q111/Q7) knock-in mice. Wild-type expression is shown in the leftbar of each pair and knock-in expression in the right bar of each pair.ZFP-TFs comprising the specified ZFP fused to the KRAB repression domainwere tested using three different concentrations of ZFP mRNA in thetransfections. FIG. 3E depicts mutant Htt repression by CAG-targetedZFPs in a HD patient derived fibroblast line (CAG15/70) (SEQ ID NOS 239and 240). In this fibroblast line, the wild type Htt allele comprises 15CAG repeats (“099T(CAG15)” (SEQ ID NO: 239), middle bar of eachindicated condition) and the mutant expanded Htt allele comprises 70 CAGrepeats (“099C(CAG70)” (SEQ ID NO: 240), right bar of each indicatedcondition). FIG. 3F shows selective repression of mutant Htt expressionin 4 different HD patient derived fibroblast cell lines. The numbersabove each grouping indicate the number of CAG repeats on the wildtypeHtt allele (e.g. 15 (SEQ ID NO: 239) or 18 (SEQ ID NO: 245)) and on themutant allele (e.g. 70 (SEQ ID NO: 240), 67 (SEQ ID NO: 242), 45 (SEQ IDNO: 243) and 44 (SEQ ID NO: 244)); where two different doses of ZFP mRNAwere tested. The left-bar of each pair shows wild-type Htt expressionand the right bar of each shows expression of mutant Htt. FIG. 3Gdepicts Htt expression in HD derived patient fibroblasts as assayed byWestern blot analysis in the presence of the ZFP-TFs 30640, 32528 and30657. The slower migrating protein bands are those produced by theexpanded mutant Htt alleles. 32528 binds to the transcription start siteof Htt (TSS) and thus inhibits expression from both alleles, while 30640and 30657 bind to the CAG repeats (CAG). FIG. 3G discloses “CAG 15/67”as SEQ ID NOS 239 and 242, and “CAG 15/70” as SEQ ID NOS 239 and 240.

FIGS. 4A and 4B depict repression of the mutant Htt in a HD patientderived fibroblast line by a panel of ZFPs targeted to the CAG repeat. Arange of RNA concentrations were used from 0.1 ng to 3 μg. In FIGS. 4Aand 4B, the left bar of each indicated conditions shows total Httexpression, the middle bar shows expression of Htt in fibroblasts inwhich the Htt allele comprises 18 CAG repeats (“099T(CAG18)” (SEQ ID NO:245) and the mutant expanded Htt allele comprises 45 CAG repeats 099T(CAG45) (SEQ ID NO: 243). FIG. 4B discloses “CAG15” as SEQ ID NO: 239and “CAG70” as SEQ ID NO: 240.

FIG. 5 shows the effect of CAG-targeted ZFP repressors on the expressionof Htt and other CAG-containing genes in HD patient-derived fibroblasts.The left bar under each indicated condition shows results with 30640;the middle bar under each indicated condition shows results with 30675;and the right bar shows mock transfections. FIG. 5 discloses CAG repeatsas SEQ ID NOS 240, 239, 253, 249, 254-258, 258-259, and 259,respectively, in order of appearance.

FIGS. 6A and 6B depict an experiment that examines the genome-widespecificity of three CAG-targeted ZFPs. FIG. 6A depicts qPCR analysis ofHtt repression performed on six biological replicates (six separatetransfections) of HD fibroblasts (CAG18 (SEQ ID NO: 245) (middlebars)/CAG45 (SEQ ID NO: 243), right bars) using 30640, 30645, or 33074.The four most similar replicates by qPCR were then selected formicroarray analysis, and the data is presented in FIG. 6B.

FIG. 7 depicts Htt repression in CAG17/69 (SEQ ID NOS 236 and 246)neuronal stem cells (NSC). The cells were transfected with ZFP mRNA atindicated doses. Left bars under each of the indicated doses showresults in CAG17 (SEQ ID NO: 236) cells, middle bars show results inwild-type cells, right bars show results in CAG69 (SEQ ID NO: 246)cells.

FIG. 8 depicts Htt expression in neurons differentiated from HDembryonic stem cells (ESC) (CAG 17/48) (SEQ ID NOS 236 and 247) treatedwith ZFP TFs. The cells were transfected with ZFP mRNA at indicateddoses.

FIG. 9 depicts repression of a mutant Htt transgene expression in R6/2mice following treatment with the ZFP TF 30640.

FIGS. 10A through 10D depict ZFPs with multimerization domains thatspecifically targets expanded CAG repeats, as illustrated in FIG. 1D.FIG. 10A shows a single ZFP that have four components: (i) a KOXrepressor domain (oval labeled “repressor”); (ii) an array of 2-6fingers (two shown, small ovals marked “Z”) that binds to (CAG)_(N) or apermutation of this sequence; and (iii) two dimerization domains(rectangles labeled “d1” and “d2”) that interact in an antiparallelconfiguration. These domains allow the ZFP to polymerize within themajor groove of a CAG tract. FIG. 10B shows a sketch of the bindingevent with a multimer of 3 ZFPs. It will be apparent that any number ofmultimers can be used and that the functional domain may be positionedanywhere on one or more of the individual ZFPs and that these diagramsare applicable to TALE-TFs as well. FIG. 10B discloses the sequences asSEQ ID NO: 260. FIG. 10C shows protein sequences of the four ZFP monomerscaffolds that are designed to multimerize via interactions betweendimerizing zinc fingers (DZ). Scaffolds are named DZ1 (SEQ ID NOS 180 &213), DZ2 (SEQ ID NOS 181 & 214), DZ3 (SEQ ID NOS 182 & 215) and DZ4(SEQ ID NOS 183 & 216). Dimerizing zinc finger domains are underlined,while the repression domain and nuclear localization sequence areindicated by bold underline and italic text (respectively). FIG. 10Dshows protein sequences of the seven ZFP monomer scaffolds that aredesigned to multimerize via interactions between coiled-coils (CC).Scaffolds are named CC1 (SEQ ID NOS 184 & 217), CC2 (SEQ ID NOS 185 &218), CC3 (SEQ ID NOS 186 & 219), CC4 (SEQ ID NO S 187 & 220), CC2 (SEQID NOS 221 & 222), CC5 (SEQ ID NOS 188 & 223), CC6 (SEQ ID NOS 189 &224) and CC7 (SEQ ID NOS 190 & 225). Coiled-coil sequences areunderlined, while the repression domain and nuclear localizationsequence are indicated by bold underline and italic text (respectively).The location of the ZFP region of each scaffold, which will vary betweendesigns, is indicated by “[ZFP].” The location of the (DNA-binding) ZFPregion of each scaffold, which will vary between designs, is indicatedby “[ZFP].”

FIGS. 11A and 11B depict activity of ZFP-TFs with dimerization domains.In FIG. 11A, ZFP-TFs with “coiled coil” (CC) domains were tested withluciferase reporters. pRL-Htt CAG17 (“CAG17” disclosed as SEQ ID NO:236) (left bar of each pair) stands for renilla luciferase reportercontrolled by human Htt promoter/exon1 fragment with 17 CAG (SEQ ID NO:236); pGL3-Htt-CAG47 (“CAG47” disclosed as SEQ ID NO: 235) (right bar ofeach pair) stands for firefly luciferase reporter controlled by humanHtt promoter/exon1 fragment with 47 CAG repeats (SEQ ID NO: 235). Seetext in Example 10 for description of the various dimerization domains.In FIG. 11B, ZFPs with the dimerizing zinc finger “DZ” domains weretested with the same luciferase reporters, and demonstrates increasedrepression with some ZFP-TF dimerization domains. The left bar in eachdoublet indicates the expression from the 17CAG repeat (SEQ ID NO: 236)Htt allele while the right bar indicates expression from 47 CAG repeat(SEQ ID NO: 235) Htt allele.

FIGS. 12A and 12B depict repression of Htt by ZFP-ZFP-KOX proteins. FIG.12A depicts Htt repression by the single 33088 and 33084 ZFP-TFs, andrepression by the 33088-33088 and 33088-33084 ZFP-ZFP-KOX proteins inwild-type (left bar), CAG18 (SEQ ID NO: 245) (middle bar) and CAG45 (SEQID NO: 243) (right bar) (FIG. 12A) HD fibroblasts; FIG. 12B depicts Httrepression by 33088-33088 and 33088-33084 ZFP-ZFP-KOX in wild type (leftbar), CAG 20 (SEQ ID NO: 248) (middle bar) and CAG41 (SEQ ID NO: 249)(left bar) HD fibroblasts.

FIGS. 13A through 13E depict activation of mouse Htt. FIG. 13Ademonstrates ZFP-TF-driven up-regulation of the mouse Htt genes at theRNA level in Neuro2A cells using a ZFP fused to the p65 activationdomain. Double bars indicate duplicate transfections. FIG. 13B depicts aWestern blot demonstrating increased Htt protein production driven bythe ZFP. FIG. 13C depicts a wild type mouse Htt allele and a “knock in”Htt allele where mouse sequence (most of exon1 and part of intron 1,line above wild-type allele schematic) has been replaced withcorresponding human sequence with CAG expansion (line over knock-inallele schematic). FIG. 13C discloses “CAG7” as SEQ ID NO: 261 and“CAG>100” as SEQ ID NO: 241. FIG. 13D depicts the alignment betweenmouse sequence (SEQ ID NO:191) that was replaced with the correspondinghuman sequence (SEQ ID NO:192) such that the knock-in allele hassufficient sequence divergence to allow ZFPs (shown in A and B) to bedesigned to bind specifically the mouse sequence. FIG. 13E depictsspecific activation of the wild type mouse Htt allele in immortalizedstriatal cells derived from the HdhQ111/Q7 knock-in mice. The left-barshows results in wild-type cells and right bar shows results in knock-inmutant allele cells.

FIGS. 14A and 14B depict the results of Cel-I mismatch assays(Surveyor™, Transgenomics) following treatment of K562 cells with Httspecific ZFN pairs. The percent NHEJ activity (in-del) for an active ZFNis shown at the bottom of the corresponding lane. “GFP” indicates cellsthat have been transfected with a GFP encoding plasmid. FIG. 14A depictsresults from ZFNs that cleave early Htt exons while FIG. 14B depictsresults from ZFNs that cleave near the stop codon. Inactive ZFN pairswere also observed (lanes not annotated with in-del percentages).

FIG. 15 depicts graphs of the Htt repression results for severalcandidate TALE-TF proteins. The TALE-TFs were tested in HD patientderived fibroblasts (CAG 20/41 (SEQ ID NOS 248 and 246)). The resultsdemonstrate that some of the TALE TFs were active in repressing overallHtt expression, while others exhibited mutant Htt-preferentialrepression.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for treating Huntington'sdisease (HD). In particular, Htt-modulating transcription factorscomprising zinc finger proteins (ZFP TFs) or TALEs (TALE-TF) and methodsutilizing such proteins are provided for use in treating or preventingHuntington's disease. For example, ZFP-TFs or TALE-TFs which repressexpression of a mutant Htt allele or activate expression of a wild-typeHtt allele are provided. In addition, zinc finger nucleases (ZFNs), TALEnucleases (TALENs) or CRISPR/Cas nuclease systems that modify thegenomic structure of the genes associated with HD are provided. Forexample, ZFNs, TALENs or CRISPR/Cas nuclease systems that are able tospecifically alter portions of a mutant form of Htt are provided. Theseinclude compositions and methods using engineered zinc finger proteinsor engineered TALE proteins, i.e., non-naturally occurring proteinswhich bind to a predetermined nucleic acid target sequence.

Thus, the methods and compositions described herein provide methods fortreatment and prevention of Huntington's Disease, and these methods andcompositions can comprise zinc finger transcription factors or TALEtranscription factors that are capable of modulating target genes aswell as engineered zinc finger and TALE nucleases and CRISPR/Casnuclease systems capable of modifying or editing Htt.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL,Second edition, Cold Spring Harbor Laboratory Press, 1989 and Thirdedition, 2001; Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, New York, 1987 and periodic updates; the seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger binding domains or TALE DNA binding domains can be“engineered” to bind to a predetermined nucleotide sequence, for examplevia engineering (altering one or more amino acids) of the recognitionhelix region of a naturally occurring zinc finger protein or byengineering the RVDs of a TALE protein. Therefore, engineered zincfinger proteins or TALEs are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering zinc finger proteins orTALEs are design and selection. A designed zinc finger protein or TALEis a protein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; and 6,534,261; see also International Patent Publication Nos.WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 andU.S. Patent Publication No. 2011/0301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759;International Patent Publication Nos. WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197, WO02/099084; and WO 2011/146121; and U.S. Patent Publication No.2011/0301073.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALE proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and−cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2008/0131962;and 2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogeneous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “multimerization domain”, (also referred to as a “dimerization domain”or “protein interaction domain”) is a domain incorporated at the amino,carboxy or amino and carboxy terminal regions of a ZFP TF or TALE TF.These domains allow for multimerization of multiple ZFP TF or TALE TFunits such that larger tracts of trinucleotide repeat domains becomepreferentially bound by multimerized ZFP TFs or TALE TFs relative toshorter tracts with wild-type numbers of lengths. Examples ofmultimerization domains include leucine zippers. Multimerization domainsmay also be regulated by small molecules wherein the multimerizationdomain assumes a proper conformation to allow for interaction withanother multimerization domain only in the presence of a small moleculeor external ligand. In this way, exogenous ligands can be used toregulate the activity of these domains.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALE protein asdescribed herein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to upregulate gene expression. ZFPs fusedto domains capable of regulating gene expression are collectivelyreferred to as “ZFP-TFs” or “zinc finger transcription factors”, whileTALEs fused to domains capable of regulating gene expression arecollectively referred to as “TALE-TFs” or “TALE transcription factors.”When a fusion polypeptide in which a ZFP DNA-binding domain is fused toa cleavage domain (a “ZFN” or “zinc finger nuclease”), the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site. When a fusionpolypeptide in which a TALE DNA-binding domain is fused to a cleavagedomain (a “TALEN” or “TALE nuclease”), the TALE DNA-binding domain andthe cleavage domain are in operative linkage if, in the fusionpolypeptide, the TALE DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the cleavage domain is ableto cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel, et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields, et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and International PatentPublication No. WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

DNA-Binding Domains

Described herein are compositions comprising a DNA-binding domain thatspecifically bind to a target sequence in any gene comprising atrinucleotide repeat, including, but not limited to, Htt. AnyDNA-binding domain can be used in the compositions and methods disclosedherein.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, etal. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) NatureBiotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancementof binding specificity for zinc finger binding domains has beendescribed, for example, in co-owned International Patent Publication No.WO 02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-ownedInternational Patent Publication No. WO 02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; and 6,200,759; International Patent Publication Nos. WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536; and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In certain embodiments, the DNA binding domain is an engineered zincfinger protein that binds (in a sequence-specific manner) to a targetsite in a Htt gene and modulates expression of Htt. The ZFPs can bindselectively to either a mutant Htt allele or a wild-type Htt sequence.Htt target sites typically include at least one zinc finger but caninclude a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6 or morefingers). Usually, the ZFPs include at least three fingers. Certain ofthe ZFPs include four, five or six fingers, while some ZFPs include 8,9, 10, 11 or 12 fingers. The ZFPs that include three fingers typicallyrecognize a target site that includes 9 or 10 nucleotides; ZFPs thatinclude four fingers typically recognize a target site that includes 12to 14 nucleotides; while ZFPs having six fingers can recognize targetsites that include 18 to 21 nucleotides. The ZFPs can also be fusionproteins that include one or more regulatory domains, which domains canbe transcriptional activation or repression domains. In someembodiments, the fusion protein comprises two ZFP DNA binding domainslinked together. These zinc finger proteins can thus comprise 8, 9, 10,11, 12 or more fingers. In some embodiments, the two DNA binding domainsare linked via an extendable flexible linker such that one DNA bindingdomain comprises 4, 5, or 6 zinc fingers and the second DNA bindingdomain comprises an additional 4, 5, or 5 zinc fingers. In someembodiments, the linker is a standard inter-finger linker such that thefinger array comprises one DNA binding domain comprising 8, 9, 10, 11 or12 or more fingers. In other embodiments, the linker is an atypicallinker such as a flexible linker. The DNA binding domains are fused toat least one regulatory domain and can be thought of as a7FP-ZFP-TF′architecture. Specific examples of these embodiments can bereferred to as “ZFP-ZFP-KOX” which comprises two DNA binding domainslinked with a flexible linker and fused to a KOX repressor and“ZFP-KOX-ZFP-KOX” where two ZFP-KOX fusion proteins are fused togethervia a linker.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort, et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon, et al. (1989) Gene82:115-118; Perler, et al. (1994) Nucleic Acids Res. 22:1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble, et al. (1996)J. Mol. Biol.263:163-180; Argast, et al. (1998)J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier, et al. (2002)Molec. Cell 10:895-905; Epinat, et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth, et al. (2006) Nature 441:656-659; Pâques, et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.2007/0117128.

“Two handed” zinc finger proteins are those proteins in which twoclusters of zinc finger DNA binding domains are separated by interveningamino acids so that the two zinc finger domains bind to twodiscontinuous target sites. An example of a two handed type of zincfinger binding protein is SIP1, where a cluster of four zinc fingers islocated at the amino terminus of the protein and a cluster of threefingers is located at the carboxyl terminus (see Remade, et al. (1999)EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in theseproteins is able to bind to a unique target sequence and the spacingbetween the two target sequences can comprise many nucleotides.Two-handed ZFPs may include a functional domain, for example fused toone or both of the ZFPs. Thus, it will be apparent that the functionaldomain may be attached to the exterior of one or both ZFPs (see, FIG.1C) or may be positioned between the ZFPs (attached to both ZFPs) (see,FIG. 4).

Specific examples of Htt-targeted ZFPs are disclosed in Tables 1A and1B. The first column in this table is an internal reference name(number) for a ZFP and corresponds to the same name in column 1 ofTables 2A and 2B. “F” refers to the finger and the number following “F”refers which zinc finger (e.g., “F1” refers to finger 1).

TABLE 1A Htt-targeted zinc finger proteins SBS Design # F1 F2 F3 F4 F5F6 18856 RSDDLSR RNDNRTK RSDDLTR RSDDRKT RSADLTR QSSDLRR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: 2) NO: 3) NO: 4) NO: 5)NO: 6) 1) 25920 RSAALSR RSDALAR RSDNLSE KRCNLRC QSSDLRR NA (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 58) 59) 60) 61) 6)25921 WRSCRSA DRSNLSR QRTHLTQ RSAHLSR TSGHLSR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: 62) 9) 53) 46) 43) 25923 RSDDLSRRNDNRTK WRSCRSA RSDNLAR QSGHLSR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: 1) 2) 62) 7) 41) 25922 RSAALSR RSDALARRSDNLSE KRCNLRC QSSDLSR DRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 58) 59) 60) 61) 31) 13)

TABLE 1B Human and Mouse Htt-targeted zinc finger proteins SBS Design #F1 F2 F3 F4 F5 F6 32468 RSDNLAR WRGDRVK DRSNLSR TSGSLTR ERGTLAR RSDDRKT(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO:7) 8) 9) 10) 11) 4) 32501 RSDALSR DRSHLAR RSDHLSR QSSDLTR TSGNLTRDRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO:NO: NO: 12) 13) 14) 15) 16) 13) 31809 RSDDLSR RNDNRTK RSDDLTR RSDDRKTRSDDLTR QSSDLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 1) 2) 3) 4) 3) 6) 32528 QSGHLQR TSGNLTR QSGDLTR DRSHLARRSDVLST VRSRLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 17) 16) 18) 13) 19) 20) 30580 RSDNLAR WRGDRVK DRSDLSRRSDALAR ERGTLAR RSDDRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 7) 8) 22) 59) 11) 4) 30929 DRSTLRQ DRSDLSRQSSTRAR RSDTLSE HRRSRWG NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:NO: NO: NO: NO: 21) 22) 23) 24) 25) 32538 DRSDLSR RRDTLRS RSDHLSTQSAHRIT QSGDLTR DRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 22) 26) 27) 28) 18) 13) 32567 RSDHLSE QNAHRKTQSSDLSR HRSTRNR QSSDLSR HRSTRNR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 29) 30) 31) 32) 31) 32) 29627 DRSNLSRLRQDLKR DRSHLTR DRSNLTR RSDHLST QSAHRIT (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 9) 33) 34) 35) 27) 28) 29628TSGNLTR LKQMLAV RSDSLSA DRSDLSR RSDALST DRSTRTK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 16) 36) 37) 22) 38) 39)29631 QSSDLSR DRSALAR QSSDLSR QSGHLSR RSDVLSE TSGHLSR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 31) 40) 31) 41)42) 43) 29632 RSDTLSE KLCNRKC TSGNLTR HRTSLTD RSAHLSR QSGNLAR (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 24) 44)16) 45) 46) 47) 29637 DRSNLSR QSGNLAR DRSNLSR LKHHLTD QSGDLTR YRWLRNN(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO:9) 47) 9) 48) 18) 49) 29638 RSDHLSQ RSAVRKN QSSDLSR QSGDLTR WSTSLRA NA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 50) 51) 31)18) 52) 25917 DRSNLSR QRTHLTQ RSSHLSR TSGSLSR TRQNRDT NA (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 9) 53) 54) 55) 56) 25916DQSTLRN RSAALSR RSDALAR RSDNLSE KRCNLRC NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: 57) 58) 59) 60) 61) 33074 RSDNLSEKRCNLRC QSGDLTR QSGDLTR RSDNLSE KRCNLRC (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 60) 61) 18) 18) 60) 61) 33080QSGDLTR QSGDLTR RSDNLSE KRCNLRC QSGDLTR QSGDLTR (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 18) 18) 60) 61) 18) 18)33084 QSSDLSR HRSTRNR RSDTLSE RRWTLVG NA NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: 31) 32) 24) 64) 33088 QSSDLSR HRSTRNR RSAVLSEQSSDLSR HRSTRNR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 31) 32) 148) 31) 32) 30643 RSDNLSE KRCNLRC QSSDLSR QWSTRKRQSSDLSR QWSTRKR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 60) 61) 31) 63) 31) 63) 30648 RSDNLSE KRCNLRC RSDNLSEKRCNLRC RSDNLSE KRCNLRC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 60) 61) 60) 61) 60) 61) 30645 RSDNLSE KRCNLRCQSSDLSR QWSTRKR QSGDLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:NO: NO: NO: NO: 60) 61) 31) 63) 18) 30640 QSSDLSR QWSTRKR QSSDLSRQWSTRKR QSGDLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 31) 63) 31) 63) 18) 30657 RSDTLSE RRWTLVG QSSDLSR HRSTRNRQSSDLSR HRSTRNR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 24) 64) 31) 32) 31) 32) 30642 QSGDLTR QSSDLSR QWSTRKRQSSDLSR QWSTRKR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 18) 31) 63) 31) 63) 30646 RSDNLSE KRCNLRC QSGDLTR QSSDLSRQWSTRKR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO:60) 61) 18) 31) 63) 32220 RSDVLSE QSSDLSR HRSTRNR NA (SEQ ID (SEQ ID(SEQ ID NA NA NO: NO: NO: 42) 31) 32) 32210 QSGDLTR QSSDLSR QWSTRKR NA(SEQ ID (SEQ ID (SEQ ID NA NA NO: NO: NO: 18) 31) 63) 32215 RSDNLRERSDNLSE KRCNLRC NA (SEQ ID (SEQ ID (SEQ ID NA NA NO: NO: NO: 65) 60) 61)30658 QSSDLSR HRSTRNR QSSDLSR HRSTRNR QSSDLSR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 31) NO: 32) NO: 31) NO: 32) NO: 31) 32218 QSSDLSRQSSDLSR NA NA NA NA (SEQ ID (SEQ ID NO: NO: 31) 31) 32427 ERGTLARTSGSLTR RSDNLAR DPSNRVG RSDDLSK DNSNRIK (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 11) NO: 10) NO: 7) NO: 78) NO: 149) NO: 150) 32653RSDHLSE QSGHLSR RSDDLTR YRWLLRS QSSDLSR RKDALVA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 29) 41) 3) 66) 31) 67)32677 QSGDLTR RRADLSR DRSHLTR DRSHLAR DRSNLSR LAQPRNK (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 18) 68) 34) 13)9) 69) 33560 ERGTLAR QSGSLTR RSDNLAR DDSHRKD RSDDLSK DNSNRIK (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 11) NO: 84) NO: 7) NO: 151)NO: 149) NO: 150) 33583 DRSNLSR HKQHRDA DRSDLSR RRTDLRR RSANLAR DRSHLAR(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 9) NO: 76) NO: 22)NO: 77) NO: 73) NO: 13) 32685 RSDHLSA RSADRTR RSDVLSE TSGHLSR RSDDLTRTSSDRKK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO:NO: NO: 70) 71) 42) 43) 3) 72) 32422 RSANLAR RSDDLTR RSDTLSE HHSARRCERGTLAR DRSNLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 73) 3) 24) 74) 11) 35) 32428 RSDVLST DNSSRTR DRSNLSRHKQHRDA DRSDLSR RRTDLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 19) 75) 9) 76) 22) 77) 32430 RSDVLST VRSRLRRERGTLAR TSGSLTR RSDNLAR DPSNRVG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 19) 20) 11) 10) 7) 78) 32432 RSDVLSTVRSRLRR ERGTLAR TSGSLTR RSDHLSA RSADLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 19) 20) 11) 10) 70) 79) 32714RSDVLST DNSSRTR ERGTLAR QSGNLAR DRSHLTR RNDDRKK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 19) 75) 11) 47) 34) 80)32733 DRSNLSR QKVTLAA RSAHLSR TSGNLTR DRSDLSR RRSTLRS (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 9) 81) 46) 16)22) 82) 30901 DRSALSR QSGSLTR QSSDLSR LKWNLRT RSDNLAR LKWDRQT (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 83) 84)31) 85) 7) 86) 31952 QSGALAR RSDDLTR DRSALSR RSDHLTQ QSGDLTR WSTSLRA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 147) NO: 3) NO: 83)NO: 152) NO: 18) NO: 52) 31921 RSDSLLR RSDDLTR QSGDLTR RRDWLPQ DRSNLSRRSDDRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 153) NO: 3)NO: 18) NO: 154) NO: 9) NO: 4) 30906 DRSHLSR TSGNLTR QSGDLTR DRSHLARRSDVLST VRSRLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 87) 16) 18) 13) 19) 20)

The sequence and location for the target sites of these proteins aredisclosed in Tables 2A and 2B. Tables 2A and 2B show the targetsequences for the indicated zinc finger proteins. Nucleotides in thetarget site that are contacted by the ZFP recognition helices areindicated in uppercase letters; non-contacted nucleotides indicated inlowercase.

TABLE 2A Target sites on human and mouse Htt SBS # Target Site 18856AcGCTGCGCCGGCGGAGGCGgggccgcg (SEQ ID NO: 88) 25920gcGCTCAGCAGGTGGTGaccttgtggac_ (SEQ ID NO: 103) 25921atGGTGGGAGAGACTGTgaggcggcagc_ (SEQ ID NO: 104) 25923tgGGAGAGacTGTGAGGCGgcagctggg (SEQ ID NO: 105) 25922atGGCGCTCAGCAGGTGGTGaccttgtg_ (SEQ ID NO: 106)

TABLE 2B Target sites on human and mouse Htt SBS # Target Site 32468agCCGGCCGTGGACTCTGAGccgaggtg_ (SEQ ID NO: 89) 32427cgCACTCGcCGCGAGgGTTGCCgggacg_ (SEQ ID NO: 155) 32501gtGGCGATGCGGGGGGCGTGgtgaggta_ (SEQ ID NO: 90) 31809acGCTGCGCCGGCGGAGGCGgggccgcg_ (SEQ ID NO: 88) 32528ccGGGACGGGTCCAaGATGGAcggccgc_ (SEQ ID NO: 91) 30580agCCGGCCGTGGACTCTGAGccgaggtg_ (SEQ ID NO: 89) 30929ccGTCCCGGCAGCCCCCacggcgccttg_ (SEQ ID NO: 92) 30658ctGCTGCTGCTGCTGCTgctggaaggac_ (SEQ ID NO: 108) 32538cgGGTCCAAGATGGACGGCCgctcaggt_ (SEQ ID NO: 93) 32567ctGCTGCTGCTGCTGGAAGGacttgagg_ (SEQ ID NO: 94) 29627tcAGATGGGACGGCGCTGACctggctgg_ (SEQ ID NO: 95) 29628ctGCCATGGACCTGAATGATgggaccca_ (SEQ ID NO: 96) 29631gtGGTCTGGGAGCTGTCGCTgatgggcg_ (SEQ ID NO: 97) 29632ccGAAGGGCCTGATtCAGCTGttacccc_ (SEQ ID NO: 98) 29637aaCTTGCAAGTAACaGAAGACtcatcct_ (SEQ ID NO: 99) 29638ctTGTACAGCTGTGAGGgtgagcataat_ (SEQ ID NO: 100) 25917gcCATGGTGGGAGAGACtgtgaggcggc_ (SEQ ID NO: 101) 25916ctCAGCAGGTGGTGACCttgtggacatt_ (SEQ ID NO: 102) 33074agCAGCAGcaGCAGCAgCAGCAGcagca_ (SEQ ID NO: 157) 33080caGCAGCAgCAGCAGcaGCAGCAgcagc_ (SEQ ID NO: 107) 33084tgCTGCTGctGCTGCTgctgctggaagg_ (SEQ ID NO: 109) 33088ctGCTGCT9CTGctGCTGCTgctggaag_ (SEQ ID NO: 158) 30643caGCAGCAGCAGCAgCAGCAGcagcagc_ (SEQ ID NO: 107) 30648agCAGCAGCAGCAGCAGCAGcagcagca_ (SEQ ID NO: 157) 30645caGCAGCAGCAgCAGCAGcagcagcagc_ (SEQ ID NO: 107) 30640caGCAGCAGCAGCAGCAgcagcagcagc_ (SEQ ID NO: 107) 30657ctGCTGCTGCTGCTgCTGCTGgaaggac_ (SEQ ID NO: 108) 30642caGCAGCAGCAGCAGCAgcagcagcagc_ (SEQ ID NO: 107) 30646caGCAGCAGCAgCAGCAGcagcagcagc_ (SEQ ID NO: 107) 32220ctGCTGCT9CTGctgctgctgctggaagg_ (SEQ ID NO: 208) 32210caGCAGCAGCAgcagcagcagcagcagc_ (SEQ ID NO: 107) 32215agCAGCAGCAGcagcagcagcagcagca (SEQ ID NO: 110) 32218tGCTGCTgctgctgctgctgctggaagg (SEQ ID NO: 111) 32653ggCTGGCTTTTGCGGGAAGGggcggggc (SEQ ID NO: 112) 32677gaATTGACaGGCGGAtGCGTCGtcctct_ (SEQ ID NO: 113) 33560cgCACTCGcCGCGAGgGTTGCCgggacg_ (SEQ ID NO: 155) 33583gcGGCGAGtGCGTCCCGTGACgtcatgc_ (SEQ ID NO:209) 32685atTCTGCGGGTCTGGCGTGGcctcgtct_ (SEQ ID NO: 114) 32422gtGACGTCATGCCGGCGGAGacgaggcc_ (SEQ ID NO: 115) 32428gtGCGTCCCGTGACGTCATGccggcgga_ (SEQ ID NO: 116) 32430gcCGCGAGgGTTGCCGGGACGggcccaa_ (SEQ ID NO: 117) 32432ccGCGAGGGTTGCCGGGACGggcccaag_ (SEQ ID NO: 118) 32714caTCGGGCagGAAGCCGTCATGgcaacc_ (SEQ ID NO: 119) 32733tcCTGCCCGATGGGACAGACcctgaaga_ (SEQ ID NO: 120) 30901gtACTGAGcAATGCTGTAGTCagcaatc_ (SEQ ID NO: 121) 31952ccTGTCCAgAGGGTCGCGGTAcctccct_ (SEQ ID NO: 159) 31921tgCCGGACCTGGCAGCGGCGgtggtggc_ (SEQ ID NO: 160) 30906ccGGGACGGGTCCAaGATGGAcggccgc_ (SEQ ID NO: 91)

In certain embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector (TALE)DNA binding domain. See, e.g., U.S. Patent Publication No. 2011/0301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay, et al. (2007) Science 318:648-651). These proteins contain aDNA binding domain and a transcriptional activation domain. One of themost well characterized TALEs is AvrBs3 from Xanthomonas campestgris pv.Vesicatoria (see Bonas, et al. (1989) Mol Gen Genet 218:127-136 andInternational Patent Publication No. WO 2010/079430). TALEs contain acentralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack, et al. (2006) J Plant Physiol163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer, et al. (2007) Appl and Envir Micro 73(13):4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

Specificity of these TALEs depends on the sequences found in the tandemrepeats. The repeated sequence comprises approximately 102 bp and therepeats are typically 91-100% homologous with each other (Bonas, et al.,ibid). Polymorphism of the repeats is usually located at positions 12and 13 and there appears to be a one-to-one correspondence between theidentity of the hypervariable diresidues at positions 12 and 13 with theidentity of the contiguous nucleotides in the TALE's target sequence(see Moscou and Bogdanove (2009) Science 326:1501 and Boch, et al.(2009) Science 326:1509-1512). Experimentally, the code for DNArecognition of these TALEs has been determined such that an HD sequenceat positions 12 and 13 leads to a binding to cytosine (C), NG binds toT, NI to A, C, G or T, NN binds to A or G, and IG binds to T. These DNAbinding repeats have been assembled into proteins with new combinationsand numbers of repeats, to make artificial transcription factors thatare able to interact with new sequences and activate the expression of anon-endogenous reporter gene in plant cells (Boch, et al., ibid).Engineered TAL proteins have been linked to a Fok I cleavage half domainto yield a TAL effector domain nuclease fusion (TALEN) exhibitingactivity in a yeast reporter assay (plasmid based target). SeeChristian, et al. (2010) Genetics epub 10.1534/genetics. 110.120717.See, also, U.S. Patent Publication No. 2011/0301073, incorporated byreference in its entirety.

Specific examples of designed dimerization domains to be used with ZFPsor TALE proteins are listed in Table 3. The amino acid sequences of twotypes of domain, coiled-coil (CC) and dimerizing zinc finger (DZ) arelisted.

TABLE 3 Designed dimerization domains Design name Amino acid sequenceDZ1 TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 122) TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 122) DZ2FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPV GLFVHMARNAH (SEQ ID NO: 123)TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRY EFSSHIVRGEH (SEQ ID NO: 122)DZ3 FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPVGLFVHMARNAH (SEQ ID NO: 123) HHCQHCDMYFADNILYTIHMGCHGYENPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 124) DZ4HHCQHCDMYFADNILYTIHMGCHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHGEKPTKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 125)FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPVGLFVHMARNAHGEKPFYCEHCEITFRDVVMYSLHKGYHGFRDPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 126) CC1AQLEKELQALEKKLAQLEWENQALEKELAQ (SEQ ID NO: 127)AQLKKKLQANKKELAQLKWKLQALKKKLAQ (SEQ ID NO: 128) CC2EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO: 130) CC3EQLEKKLQALEKKLAQLEWKNQALEK (SEQ ID NO: 131)ELQANKKELAQLKWELQALKKELAQ (SEQ ID NO: 132) CC4EQLEKKLQALEKKLAQLEWKNQA (SEQ ID NO: 133)QANKKELAQLKWELQALKKELAQ (SEQ ID NO: 134) CC5EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO: 130) CC6EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO: 130) CC7EQLEKKLQALEKKLAQLEWKNQALEKKLAQ (SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ (SEQ ID NO: 130)

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., ZFPs or TALEs) asdescribed herein and a heterologous regulatory (functional) domain (orfunctional fragment thereof) are also provided. Common domains include,e.g., transcription factor domains (activators, repressors,co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun,fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e.g. kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. U.S. Patent Publication Nos. 2005/0064474; 2006/0188987and 2007/0218528 for details regarding fusions of DNA-binding domainsand nuclease cleavage domains, incorporated by reference in theirentireties herein.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann, et al. (1997) J. Virol.71:5952-5962) nuclear hormone receptors (see, e.g., Torchia, et al.(1998) Curr. Opin. Cell. Biol. 10:373-383); the p65 subunit of nuclearfactor kappa B (Bitko and Barik (1998) J. Virol. 72:5610-5618 and Doyleand Hunt (1997) Neuroreport 8:2937-2942; Liu, et al. (1998) Cancer GeneTher. 5:3-28), or artificial chimeric functional domains such as VP64(Beerli, et al. (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), anddegron (Molinari, et al. (1999) EMBO J. 18:6439-6447). Additionalexemplary activation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1(Seipel, et al. (1992) EMBO J. 11:4961-4968 as well as p300, CBP, PCAF,SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr, et al. (2000)Mol. Endocrinol. 14:329-347; Collingwood, et al. (1999) J. Mol.Endocrinol. 23:255-275; Leo, et al. (2000) Gene 245:1-11;Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna, etal. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik, et al. (2000)Trends Biochem. Sci. 25:277-283; and Lemon, et al. (1999) Curr. Opin.Genet. Dev. 9:499-504. Additional exemplary activation domains include,but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8,CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa, et al.(2000) Gene 245:21-29; Okanami, et al. (1996) Genes Cells 1:87-99; Goff,et al. (1991) Genes Dev. 5:298-309; Cho, et al. (1999) Plant Mol. Biol.40:419-429; Ulmason, et al. (1999) Proc. Natl. Acad. Sci. USA96:5844-5849; Sprenger-Haussels, et al. (2000) Plant J. 22:1-8; Gong, etal. (1999) Plant Mol. Biol. 41:33-44; and Hobo, et al. (1999) Proc.Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain and a functional domain, either an activation domain or amolecule that interacts with an activation domain is suitable as afunctional domain. Essentially any molecule capable of recruiting anactivating complex and/or activating activity (such as, for example,histone acetylation) to the target gene is useful as an activatingdomain of a fusion protein. Insulator domains, localization domains, andchromatin remodeling proteins such as ISWI-containing domains and/ormethyl binding domain proteins suitable for use as functional domains infusion molecules are described, for example, in co-owned U.S. PatentPublication Nos. 2002/0115215 and 2003/0082552 and in co-ownedInternational Patent Publication No. WO 02/44376.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird, et al. (1999) Cell 99:451-454; Tyler, et al.(1999) Cell 99:443-446; Knoepfler, et al. (1999) Cell 99:447-450; andRobertson, et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem, et al. (1996) Plant Cell 8:305-321; and Wu, etal. (2000) Plant J. 22:19-27.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp, et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

In certain embodiments, the target site bound by the DNA binding domainis present in an accessible region of cellular chromatin. Accessibleregions can be determined as described, for example, in co-ownedInternational Patent Publication No. WO 01/83732. If the target site isnot present in an accessible region of cellular chromatin, one or moreaccessible regions can be generated as described in co-ownedInternational Patent Publication No. WO 01/83793. In additionalembodiments, the DNA-binding domain of a fusion molecule is capable ofbinding to cellular chromatin regardless of whether its target site isin an accessible region or not. For example, such DNA-binding domainsare capable of binding to linker DNA and/or nucleosomal DNA. Examples ofthis type of “pioneer” DNA binding domain are found in certain steroidreceptor and in hepatocyte nuclear factor 3 (HNF3). Cordingley, et al.(1987) Cell 48:261-270; Pina, et al. (1990) Cell 60:719-731; andCirillo, et al. (1998) EMBO J. 17:244-254.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-ownedInternational Patent Publication No. WO 00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. No. 6,534,261 and U.S. Patent Publication No.2002/0160940.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample U.S. Patent Publication No. 2009/0136465). Thus, the ZFP or TALEmay be operably linked to the regulatable functional domain wherein theresultant activity of the ZFP-TF or TALE-TF is controlled by theexternal ligand.

Nucleases

In certain embodiments, the fusion protein comprises a DNA-bindingbinding domain and cleavage (nuclease) domain. As such, genemodification can be achieved using a nuclease, for example an engineerednuclease. Engineered nuclease technology is based on the engineering ofnaturally occurring DNA-binding proteins. For example, engineering ofhoming endonucleases with tailored DNA-binding specificities has beendescribed. (see, Chames, et al. (2005) Nucleic Acids Res 33(20):e178;Arnould, et al. (2006) J. Mol. Biol. 355:443-458). In addition,engineering of ZFPs has also been described. See, e.g., U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and7,013,219.

In addition, ZFPs and TALEs have been fused to nuclease domains tocreate ZFNs and TALENs—functional entities that are able to recognizetheir intended nucleic acid target through their engineered (ZFP orTALE) DNA binding domains and cause the DNA to be cut near the ZFP orTALE DNA binding site via the nuclease activity. See, e.g., Kim, et al.(1996) Proc Natl Acad Sci USA 93(3):1156-1160. More recently, ZFNs havebeen used for genome modification in a variety of organisms. See, forexample, U.S. Patent Publication Nos. 2003/0232410; 2005/0208489;2005/0026157; 2005/0064474; 2006/0188987; and 2006/0063231; andInternational Patent Publication No. WO 07/014275.

Thus, the methods and compositions described herein are broadlyapplicable and may involve any nuclease of interest. Non-limitingexamples of nucleases include meganucleases, TALENs and zinc fingernucleases. The nuclease may comprise heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TALENs; meganucleaseDNA-binding domains with heterologous cleavage domains) or,alternatively, the DNA-binding domain of a naturally-occurring nucleasemay be altered to bind to a selected target site (e.g., a meganucleasethat has been engineered to bind to site different than the cognatebinding site).

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO: 250) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. Nos. 5,420,032;6,833,252; Belfort, et al. (1997) Nucleic Acids Res. 25:3379-3388;Dujon, et al. (1989) Gene 82:115-118; Perler, et al. (1994) NucleicAcids Res. 22:1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble,et al. (1996) J. Mol. Biol. 263:163-180; Argast, et al. (1998) J. Mol.Biol. 280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG (SEQ ID NO: 250) family, have been used to promotesite-specific genome modification in plants, yeast, Drosophila,mammalian cells and mice, but this approach has been limited to themodification of either homologous genes that conserve the meganucleaserecognition sequence (Monet, et al. (1999) Biochem. Biophysics. Res.Common. 255:88-93) or to pre-engineered genomes into which a recognitionsequence has been introduced (Route, et al. (1994) Mol. Cell. Biol.14:8096-106; Chilton, et al. (2003) Plant Physiology 133:956-65; Puchta,et al. (1996) Proc. Natl. Acad. Sci. USA 93:5055-60; Rong, et al. (2002)Genes Dev. 16:1568-81; Gouble, et al. (2006) J. Gene Med. 8(5):616-622).Accordingly, attempts have been made to engineer meganucleases toexhibit novel binding specificity at medically or biotechnologicallyrelevant sites (Porteus, et al. (2005) Nat. Biotechnol. 23:967-73;Sussman, et al. (2004) J. Mol. Biol. 342:31-41; Epinat, et al. (2003)Nucleic Acids Res. 31:2952-62; Chevalier, et al. (2002) Molec. Cell10:895-905; Epinat, et al. (2003) Nucleic Acids Res. 31:2952-2962;Ashworth, et al. (2006) Nature 441:656-659; Nques, et al. (2007) CurrentGene Therapy 7:49-66; U.S. Patent Publication Nos. 2007/0117128;2006/0206949; 2006/0153826; 2006/0078552; and 2004/0002092). Inaddition, naturally-occurring or engineered DNA-binding domains frommeganucleases have also been operably linked with a cleavage domain froma heterologous nuclease (e.g., Fok I).

In other embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNscomprise a zinc finger protein that has been engineered to bind to atarget site in a gene of choice and cleavage domain or a cleavagehalf-domain.

As described in detail above, zinc finger binding domains can beengineered to bind to a sequence of choice. See, for example, Beerli, etal. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev.Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol.19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637;Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineeredzinc finger binding domain can have a novel binding specificity,compared to a naturally-occurring zinc finger protein. Engineeringmethods include, but are not limited to, rational design and varioustypes of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancementof binding specificity for zinc finger binding domains has beendescribed, for example, in co-owned International Patent Publication No.WO 02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length (e.g., TGEKP (SEQ ID NO:135),TGGQRP (SEQ ID NO:136), TGQKP (SEQ ID NO:137), and/or TGSQKP (SEQ IDNO:138)). See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949for exemplary linker sequences 6 or more amino acids in length. Theproteins described herein may include any combination of suitablelinkers between the individual zinc fingers of the protein. See, also,U.S. Patent Publication No. 2011/0287512.

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer.” Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”), andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek, etal. (2012) Science 337:816-821; Jinek, et al. (2013) eLife 2:e00471, andSegal (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can beengineered to create a DSB at a desired target in a genome, and repairof the DSB can be influenced by the use of repair inhibitors to cause anincrease in error prone repair.

Nucleases such as ZFNs, TALENs and/or meganucleases also comprise anuclease (cleavage domain, cleavage half-domain). As noted above, thecleavage domain may be heterologous to the DNA-binding domain, forexample a zinc finger DNA-binding domain and a cleavage domain from anuclease or a meganuclease DNA-binding domain and cleavage domain from adifferent nuclease. Heterologous cleavage domains can be obtained fromany endonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort,et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn, etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150; and5,487,994; as well as Li, et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li, et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim, et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim, et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite, et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger- or TALE-Fok I fusions, two fusion proteins,each comprising a Fok I cleavage half-domain, can be used toreconstitute a catalytically active cleavage domain. Alternatively, asingle polypeptide molecule containing a zinc finger binding domain andtwo Fok I cleavage half-domains can also be used. Parameters fortargeted cleavage and targeted sequence alteration using zinc finger- orTALE-Fok I fusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPatent Publication No. WO 07/014275, incorporated herein in itsentirety. Additional restriction enzymes also contain separable bindingand cleavage domains, and these are contemplated by the presentdisclosure. See, for example, Roberts, et al. (2003) Nucleic Acids Res.31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987; and2008/0131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains. Exemplary engineered cleavagehalf-domains of Fok I that form obligate heterodimers include a pair inwhich a first cleavage half-domain includes mutations at amino acidresidues at positions 490 and 538 of Fok I and a second cleavagehalf-domain includes mutations at amino acid residues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication Nos. 2008/0131962 and 2011/0201055, the disclosure ofwhich is incorporated by reference in its entirety for all purposes. Incertain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFok I), for instance mutations that replace the wild type Gln (Q)residue at position 486 with a Glu (E) residue, the wild type Iso (I)residue at position 499 with a Leu (L) residue and the wild-type Asn (N)residue at position 496 with an Asp (D) or Glu (E) residue (alsoreferred to as a “ELD” and “ELE” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490, 538 and 537 (numbered relative to wild-type Fok I), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue, the wild type Iso (I) residue atposition 538 with a Lys (K) residue, and the wild-type His (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KKK” and “KKR” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490 and 537 (numbered relative to wild-type Fok I), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue and the wild-type His (H) residue atposition 537 with a Lys (K) residue or a Arg (R) residue (also referredto as “KIK” and “KIR” domains, respectively). (See U.S. PatentPublication No. 2011/0201055).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 2005/0064474 and 2008/0131962.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 2009/0068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector similar to those derived from the plant pathogensXanthomonas (see Boch, et al. (2009) Science 326:1509-1512 and Moscouand Bogdanove (2009) Science 326:1501) and Ralstonia (see Heuer, et al.(2007) Applied and Environmental Microbiology 73(13):4379-4384). Also,see International Patent Publication No. WO2010/079430.

Nucleases (e.g., ZFNs or TALENs) can be screened for activity prior touse, for example in a yeast-based chromosomal system as described inInternational Patent Publication No. WO 2009/042163 and U.S. PatentPublication No. 2009/0068164. Nuclease expression constructs can bereadily designed using methods known in the art. See, e.g., U.S. PatentPublication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474;2006/0188987; 2006/0063231; and International Patent Publication No. WO07/014275. Expression of the nuclease may be under the control of aconstitutive promoter or an inducible promoter, for example thegalactokinase promoter which is activated (de-repressed) in the presenceof raffinose and/or galactose and repressed in presence of glucose.

Delivery

The proteins (e.g., ZFPs, TALEs, CRISPR/Cas), polynucleotides encodingsame and compositions comprising the proteins and/or polynucleotidesdescribed herein may be delivered to a target cell by any suitable meansincluding, for example, by injection of ZFP-TF, TALE-TF proteins or byuse of ZFN or TALEN encoding mRNA. Suitable cells include but notlimited to eukaryotic and prokaryotic cells and/or cell lines.Non-limiting examples of such cells or cell lines generated from suchcells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11,CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6cells as well as insect cells such as Spodoptera fugiperda (Sf), orfungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. Incertain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cellline. Suitable cells also include stem cells such as, by way of example,embryonic stem cells, induced pluripotent stem cells, hematopoietic stemcells, neuronal stem cells and mesenchymal stem cells.

Methods of delivering proteins comprising zinc finger proteins asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

Zinc finger, TALE or CRISPR/Cas proteins as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger, TALE or CRISPR/Cas protein(s). Any vector systems may beused including, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more zinc finger or TALE protein-encoding sequences. Thus, whenone or more ZFPs, TALEs or CRISPR/Cas proteins are introduced into thecell, the sequences encoding the ZFPs, TALEs or CRISPR/Cas proteins maybe carried on the same vector or on different vectors. When multiplevectors are used, each vector may comprise a sequence encoding one ormultiple ZFPs, TALEs or CRISPR/Cas systems.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs, TALEs or CRISPR/Cassystems in cells (e.g., mammalian cells) and target tissues. Suchmethods can also be used to administer nucleic acids encoding ZFPs,TALEs or a CRISPR/Cas system to cells in vitro. In certain embodiments,nucleic acids encoding the ZFPs, TALEs or CRISPR/Cas system areadministered for in vivo or ex vivo gene therapy uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson (1992) Science256:808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani andCaskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175;Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience8:35-36; Kremer and Perricaudet (1995) British Medical Bulletin51(1):31-44; Haddada, et al., in Current Topics in Microbiology andImmunology Doerfler and Böhm (eds.) (1995); and Yu, et al. (1994) GeneTherapy 1:13-26.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,naked RNA, artificial virions, and agent-enhanced uptake of DNA.Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can alsobe used for delivery of nucleic acids. In a preferred embodiment, one ormore nucleic acids are delivered as mRNA. Also preferred is the use ofcapped mRNAs to increase translational efficiency and/or mRNA stability.Especially preferred are ARCA (anti-reverse cap analog) caps or variantsthereof. See U.S. Pat. Nos. 7,074,596 and 8,153,773, incorporated byreference herein.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, InternationalPatent Publication Nos. WO 91/17424 and WO 91/16024. Delivery can be tocells (ex vivo administration) or target tissues (in vivoadministration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal (1995) Science 270:404-410 (1995);Blaese, et al. (1995) Cancer Gene Ther. 2:291-297; Behr, et al. (1994)Bioconjugate Chem. 5:382-389; Remy, et al. (1994) Bioconjugate Chem.5:647-654; Gao, et al. (1995) Gene Therapy 2:710-722; Ahmad, et al.(1992) Cancer Res. 52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344;4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (seeMacDiarmid, et al. (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs, TALEs or CRISPR/Cas systems takeadvantage of highly evolved processes for targeting a virus to specificcells in the body and trafficking the viral payload to the nucleus.Viral vectors can be administered directly to patients (in vivo) or theycan be used to treat cells in vitro and the modified cells areadministered to patients (ex vivo). Conventional viral based systems forthe delivery of ZFPs, TALEs or CRISPR/Cas systems include, but are notlimited to, retroviral, lentivirus, adenoviral, adeno-associated,vaccinia and herpes simplex virus vectors for gene transfer. Integrationin the host genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher, et al. (1992) J. Virol.66:2731-2739; Johann, et al. (1992) J. Virol. 66:1635-1640; Sommerfelt,et al. (1990) Virol. 176:58-59; Wilson, et al. (1989) J. Virol.63:2374-2378; Miller, et al. (1991) J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West, et al. (1987) Virology160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No.WO 93/24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994)J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors aredescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin, et al. (1985) Mol. Cell. Biol. 5:3251-3260;Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat andMuzyczka (1984) PNAS 81:6466-6470; and Samulski, et al. (1989) J. Virol.63:3822-3828.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar, et al. (1995) Blood 85:3048-305; Kohn, etal. (1995) Nat. Med. 1:1017-102; Malech, et al. (1997) PNAS94(22):12133-12138). PA317/pLASN was the first therapeutic vector usedin a gene therapy trial. (Blaese, et al. (1995) Science 270:475-480).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem, et al. (1997) Immunol Immunother 44(1):10-20;Dranoff, et al. (1997) Hum. Gene Ther. 1:111-112.

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner, et al. (1998) Lancet 351(9117):1702-1703; Kearns, et al. (1996)Gene Ther. 9:748-55). Other AAV serotypes, including AAV1, AAV3, AAV4,AAV5, AAV6, AAV8AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such asAAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with thepresent invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman, et al. (1998) Hum.Gene Ther. 7:1083-1089). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker, et al.(1996) Infection 24(1):5-10; Sterman, et al. (1998) Hum. Gene Ther.9(7):1083-1089; Welsh, et al. (1995) Hum. Gene Ther. 2:205-218; Alvarez,et al. (1997) Hum. Gene Ther. 5:597-613; Topf, et al. (1998) Gene Ther.5:507-513; Sterman, et al. (1998) Hum. Gene Ther. 7:1083-1089.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han, et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751, reported that Moloney mouse leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFP,TALE or CRISPR/Cas system nucleic acid (gene. cDNA or mRNA), andre-infused back into the subject organism (e.g., patient). In apreferred embodiment, one or more nucleic acids are delivered as mRNA.Also preferred is the use of capped mRNAs to increase translationalefficiency and/or mRNA stability. Especially preferred are ARCA(anti-reverse cap analog) caps or variants thereof. See U.S. Pat. Nos.7,074,596 and 8,153,773, incorporated by reference herein in theirentireties. Various cell types suitable for ex vivo transfection arewell known to those of skill in the art (see, e.g., Freshney, et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba, et al. (1992) J.Exp. Med. 176:1693-1702).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-1 (granulocytes),and Iad (differentiated antigen presenting cells) (see Inaba, et al.(1992) J. Exp. Med. 176:1693-1702).

Stem cells that have been modified may also be used in some embodiments.For example, neuronal stem cells that have been made resistant toapoptosis may be used as therapeutic compositions where the stem cellsalso contain the ZFP TFs of the invention. Resistance to apoptosis maycome about, for example, by knocking out BAX and/or BAK using BAX- orBAK-specific TALENs or ZFNs (see, U.S. Patent Publication No.2010/0003756) in the stem cells, or those that are disrupted in acaspase, again using caspase-6 specific ZFNs for example. These cellscan be transfected with the ZFP TFs or TALE TFs that are known toregulate mutant or wild-type Htt.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory, et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull, etal. (1998) J. Virol. 72:8463-8471; Zuffery, et al. (1998) J. Virol.72:9873-9880; Follenzi, et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NS0,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

Applications

The disclosed compositions and methods can be used for any applicationin which it is desired to modulate the Htt allele, including but notlimited to, therapeutic and research applications.

Diseases and conditions which Htt repressing ZFP TFs or TALE TFs can beused as therapeutic agents include, but are not limited to, Huntington'sdisease. Additionally, methods and compositions comprising ZFNs orTALENs specific for mutant alleles of Htt can be used as a therapeuticfor the treatment of Huntington's disease.

ZFP-TFs or TALE TFs that repress a HD Htt allele may also be used inconjunction with ZFP-TFs or TALE-TFs that activate neutrotrophic factorsincluding, but not limited to, GDNF and BDNF. These ZFPs or TALEs (orpolynucleotides encoding these ZFPs or TALEs) may be administeredconcurrently (e.g., in the same pharmaceutical compositions) or may beadministered sequentially in any order.

Methods and compositions for the treatment of Huntington's disease alsoinclude stem cell compositions wherein a mutant copy of the Htt allelewithin the stem cells has been modified to a wild-type Htt allele usinga Htt-specific ZFN or TALEN.

The methods and compositions of the invention are also useful for thedesign and implementation of in vitro and in vivo models, for example,animal models of trinucleotide repeate disorders, which allows for thestudy of these disorders. Non-limiting examples of suitable in vitromodels include cells or cell lines from any organism, includingfibroblasts. Non-limiting examples of suitable animals for use as animalmodels include, invertebrates (C. elegans, drosophila), rodents (e.g.,rat or mouse), primates (e.g., non-human primates).

EXAMPLES Example 1: Design and Construction of Htt-Targeted Zinc FingerProtein Transcription Factors (ZFP-TFs) and ZFNs

Zinc finger proteins targeted to Htt were engineered essentially asdescribed in U.S. Pat. No. 6,534,261. Tables 1A and 1B show therecognition helices of the DNA binding domain of exemplary Htt-targetedZFPs, while Tables 2A and 2B show the target sequences of these ZFPs.

ZFPs with one contiguous array of zinc fingers were designed to targetsites completely within the CAG repeat region (FIG. 1B). Such ZFPs maybind longer, mutant tracts with higher affinity and/or a higher netoccupancy, achieving selective repression of the mutant allele. ZFNswere also designed that targeted sites which lay partially or whollyoutside of the CAG region (FIG. 1A), and which will therefore bind tothe wild type and mutant allele equally, and regulate expression fromboth alleles with similar efficiency. When designing zinc fingerproteins to recognize the CAG region, a set of one- and two-fingermodules can be employed in a ‘mix and match’ combination. Those modulesare shown below in Table 2C.

TABLE 2C Zinc finger recognition helices used in ZFP-TFstargeting CAG repeats (SEQ ID NO: Target site F2 F3 (F2 + F3) CAGCAGRSDNLSE KRCNLRC 161 CAGCAG RSDNLSE KPYNLRT 162 CAGCAG RSDNLSE RLWNRKQ163 CAGCAG RSDNLSV RRWNLRA 164 CAGCAG RSDNLSV RKWNRDS 165 CAGCAG RSDNLSENTSPLML 166 CAGCAG RSDNLSE RRYNLVK 167 CTGCTG RSDTLSE RRWTLVG 168 GCAGCAQSSDLSR QWSTRKR 169 GCAGCA RSAHLSR QSGDLTR 170 GCAGCA QSGDLTR QSGDLTR171 GCAGCA QSGDLTR QSSDLRR 172 GCTGCT QSSDLSR QSSDLRR 173 GCTGCT QSSDLSRHRSTRNR 174 AGC MACCRYA none 175 CAG RSANLRE none 176 CAG RNADRKK none177 CTG RSDVLSE none 42 CTG RSAVLSE none 148 GCA QSGDLTR none 18 GCAQSSDLRR none 6 GCA QNATRIK none 178 GCT QSSDLSR none 31 AAG RSDNLRE none65

Multimerizing ZFP TFs are also constructed as described above exceptthat the vector also contains sequences encoding 1 or more proteininteraction domains (also called dimerization or protein interactiondomains) that enable multimerization of the expressed protein along atract of trinucleotide repeats that is operably linked to the sequencesencoding the ZFP TF. See, FIG. 1D and FIG. 10. Table 3 showsdimerization domain designs that are used with ZFPs targeted to the CAGrepeat region. FIG. 10C shows protein sequences of the four ZFP monomerscaffolds that are designed to multimerize via interactions betweendimerizing zinc fingers (DZ), DZ1-DZ4. Designs are based on workdescribed in Gisecke, et al. (2006) Mol. Syst. Biol. 2:2006-2011. FIG.10D shows protein sequences of the seven ZFP monomer scaffolds that aredesigned to multimerize via interactions between coiled-coils (CC),CC1-CC7. The design of CC#1 is based on the work described in (McClain,et al. (2001) J. Am. Chem. Soc. 123:3151-3152), while CC#2, CC#3 andCC#4 are based on (Ghosh, et al. (2000) J. Am. Chem. Soc.122:5658-5659). CC and DZ domains allow the ZFP to polymerize within themajor groove of a CAG tract (depicted in FIG. 10B). By choosing a fingerarray and dimerization domains with appropriate binding properties,efficient binding will occur only to the expanded CAG tract of a diseaseallele.

ZFP-TFs were constructed as fusion proteins comprising a nuclearlocalization sequence, the engineered zinc finger DNA-binding domain(Tables 1A and 1B), targeted to the Htt allele, and a KRAB repressiondomain from the human KOX1 protein. See, FIGS. 1A, 1B and 1D. Thedesigned DNA-binding domains contain 3-6 finger modules, recognizing9-18 bp sequences (Tables 2A and 2B). Nucleotides in the target sitethat are contacted by the ZFP recognition helices are indicated inuppercase letters; non-contacted nucleotides indicated in lowercase.ZFP-ZFP-TF molecules were also constructed where two ZFP DNA bindingdomains were fused with a flexible linker and fused to a KRAB repressiondomain (FIG. 1E). DNA binding domains were chosen from Tables 2A and 2B.

Example 2: Repression of Both Alleles of Htt in Human and Mouse Cells

To repress both alleles of the Htt (non-allele-specific), ZFPs weredesigned to bind to the Htt promoter and exon 1 region, wherein thetarget site was not entirely within the CAG repeat. See, FIG. 1A. Totest the activity of the Htt repressing ZFP TFs, the ZFP TFs weretransfected into human cells and expression of Htt was monitored usingreal-time RT-PCR.

Human HEK293 cells (Graham, et al. (1977) J Gen Virol 36:59-74) werecultured in DMEM supplemented with 10% FBS and 1e⁵ cells weretransfected with 1 μg of plasmid DNA encoding indicated ZFP-KOX fusionsby Amaxa Nucleofector® following the manufacturer's instructions.

Transfected cells were incubated for 2 days, and the levels ofendogenous human Huntingtin (Htt) and normalization control beta-actin(ACTB) were analyzed by real-time PCR using Hs00918176_m1 and 4352935Eprimers and probes (Applied Biosystems), respectively, according tostandard protocols. Htt levels were expressed as Htt/ACTB ratiosnormalized to that of the mock-transfected samples (set as 1).

As shown in FIG. 2A, Htt-targeted ZFPs repressed Htt expression. Westernblot analyses were done using standard protocols to confirm thereduction in Htt protein level (FIG. 2B); p65 protein was used asloading control.

Mouse Htt-specific ZFP TFs repressors were transiently transfected intoNeuro2A cells (Klebe and Ruddle (1969) J. Cell Biol. 43:69A) using theLipofectamine® 2000 kit (Invitrogen) according to manufacturer'sprotocols. mHtt and ACTB mRNA levels were measured at 48 hours aftertransfection using ABI Taqman® primer/probe set Mm01213820 m1 and4352933E, respectively. mHtt/ACTB ratios for ZFP transfected sampleswere normalized to that of the GFP control (set as 1).

As shown in FIG. 2C, the ZFPs repressed mouse Htt expression. Inaddition, mouse Htt-specific ZFP-TF repressors can repress mouse Htt inimmortalized striatal cells, STHdh(Q111/Q7), derived from Htt knock-inmice (Trettel, et al. (2000) Hum. Mol. Genet 9:2799-2809). See, FIGS. 2Dand 2E. mRNA for indicated ZFPs were generated using the mMessagemMachine kit (Ambion), and 0.1, 0.5 or 2 ug of these mRNAs weretransfected using Amaxa nucleofector as described above. Cells wereharvested 48 hours after transfection for mHtt and ACTB expressionanalysis as described above. More significant repression in the striatalcells compared to that in Neuro2A cells was observed and may be a resultof enhanced transfection efficiency achieved via mRNA transfection instraital cells.

Example 3: Selective Repression of Mutant Htt in Human and Mouse Cells

To achieve selective repression of the mutant Htt allele, ZFPs weredesigned to bind within the CAG repeat. FIG. 1B shows one type of suchZFPs, with a contiguous array of zinc fingers linked to a repressiondomain (e.g. the KRAB domain from KOX1); these ZFPs can be designed withappropriate affinity such that threshold occupancy required fromtranscriptional repression can only be established on expanded CAGrepeats. FIGS. 1C, 1D and 1E show threeother examples of ZFP design thatcan allow specific binding to the expanded CAG repeats.

ZFPs designed as illustrated in FIG. 1B were introduced into HEK293cells and Htt expression evaluated. ZFP-encoding constructs weretransfected into HEK293 cells using FugeneHD using standard protocols.Seventy-two hours after transfection total RNA was isolated and thelevels of endogenous human Huntingtin (Htt) relative to internal controlbeta-actin (ACTB) were analyzed by real-time PCR using Hs00918176_m1 and4352935E primers and probes (Applied Biosystems), respectively. Htt/ACTBratios for ZFP transfected samples were normalized to that of the GFPcontrol (set as 1).

As shown in FIG. 3A, ZFP repressors (fused with KRAB repression domain)designed to bind to CAG repeats (FIG. 1B), either to top or bottomstrand, in HEK293 cells, effectively repressed Htt expression. FIG. 3Adepicts repressors of transcription where expression was measure induplicate transfections (separate bars in the Figure) and multiplereal-time PCR assays completed (error bars). Different levels ofrepression by individual ZFPs suggest that they have different affinityto the CAG repeat region. Because Htt alleles in HEK293 cells have 16(SEQ ID NO: 251) and 17 CAG (SEQ ID NO: 236), this result also suggeststhat “weaker” ZFPs, such as 30640, do not repress Htt alleles withwild-type (unexpanded) CAG repeat length effectively.

To test whether ZFPs such as 30640 can repress transcription of Httalleles with expanded CAG repeats, luciferase reporters controlled byHtt promoter/exon 1 fragment that contains different CAG repeat lengthswere constructed. First, the human Htt promoter/exon1 fragment wasamplified from HEK293 genomic DNA using forward primer:

(SEQ ID NO: 139) 5′ GAAGATCTCACTTGGGGTCCTCAGGTCGTGCCGACand reverse primer:

(SEQ ID NO: 140) 5′ GTATCCAAGCTTCAGCTTTTCCAGGGTCGCCTAGGCGGTCT..

The forward primer introduces a BglII site, the reverse primer changesthe first ATG of Htt into TAG and creates an AvrII site, and alsoincludes a HindIII site. The PCR product was digested with BglII andHindIII and ligated to pRL-TK vector (Promega) that was digested withthe same enzymes to generate the construct pRL-Htt. Then the human Httexon1 fragment (coding sequence minus first ATG) was amplified fromHEK293 genomic DNA or genomic DNA from HD patients with expanded CAGrepeats using forward primer:

(SEQ ID NO: 141) 5′ GCCTAGGCGACCCTGGAAAAGCTGATGAAGGCCand reverse primer: 5′

(SEQ ID NO: 142) 5′ GTATCCAAGCTTGAGCTGCAGCGGGCCCAAACTCACG.

The forward primer introduces an AvrII site, the reverse primerintroduces a HindIII site. The PCR product was digested with AvrII andHindIII and ligated to pRL-Htt vector that was digested with the sameenzymes. Clones with 10 (SEQ ID NO: 234), 17 (SEQ ID NO: 236), 23 (SEQID NO: 237) or 47 (SEQ ID NO: 235) CAG repeats (pRL-Htt-CAG(x)) wereidentified by sequencing.

pRL-Htt-CAG(x) reporters (300 ng) and pGL3-promoter reporter (100 ng,used as normalization control, Progema) were transfected into HEK293cells with or without 100 ng of ZFP 30640 expression vector. Firefly(pGL reporter) and renilla (pRL reporter) luciferase activities weremeasured 24 hours after transfection. Renilla luciferase levels werenormalized to those of firefly luciferase from the same transfectedsample, and further normalized to the renilla/firefly ratio of the“reporter only” sample.

As shown in FIG. 3B, repression of the luciferase reporters by ZFP-TF30640 increases with the length of the CAG repeat, suggesting that ZFPswith DNA binding affinities similar to that of 30640 can repress Httpromoter activity via an expanded CAG repeat, and the level ofrepression is dependent on the CAG repeat lengths.

FIG. 3C shows a similar experiment as in FIG. 3B, except the “strong”ZFP-TF 30657 was also tested, and both 30640 and 30657 were tested atmultiple doses as indicated. At every dose level, 30640 gave morerepression of the pRL-Htt-CAG47 (“CAG47” disclosed as SEQ ID NO: 235)reporter than the pRL-Htt-CAG23 reporter (“CAG23” disclosed as SEQ IDNO: 237) (CAG repeat length-dependent repression), while 30657 repressedboth reporters to similar levels. On the pRL-Htt-CAG23 (“CAG23”disclosed as SEQ ID NO: 237) reporter, 30640 gave less repression than30657 at every dose level, recapitulating the difference in theiractivities on the endogenous Htt allele with normal CAG repeat length(HEK293 cells, FIG. 3A); but on the pRL-Htt-CAG47 (“CAG47” disclosed asSEQ ID NO: 235) reporter, 30640 and 30657 gave similar repression atevery dose level, suggesting that “weaker” ZFPs such as 30640 canefficiently repress Htt promoter through an expanded CAG repeat, mostlikely because only an expanded CAG target can allow threshold occupancyrequired for repression to be established by such ZFPs.

FIG. 3D shows ZFP-TFs 30640 and 30657 (fused to the KRAB repressiondomain of KOX1) can repress the knock-in Htt allele (CAG111) inimmortalized striatal cells derived from the Hdh (Q111/Q7) knock-inmice, demonstrating the ZFPs such as 30640, which drives CAG repeatlength-dependent repression of luciferase reporters, can also repressexpression from an endogenous Htt allele that has expanded CAG repeat.mRNA for indicated ZFPs were generated using the mMessage mMachine kit(Ambion), and transfected into Hdh(Q111/Q7) cells at indicated dosesusing Amaxa nucleofector. To detect expression from the wt mouse Httallele, forward primer CAGGTCCGGCAGAGGAACC (SEQ ID NO:193) and reverseprimer TTCACACGGTCTTTCTTGGTGG (SEQ ID NO:194) were used in the real-timeRT-PCR; to detect expression from the knockin Htt allele, forward primerGCCCGGCTGTGGCTGA (SEQ ID NO:195) and reverse primerTTCACACGGTCTTTCTTGGTGG (SEQ ID NO:196) were used.

FIG. 3E show the result of testing the ZFP-TFs 30640 and 30657 in an HDpatient-derived fibroblast line (GM21756, Coriell) that has 15 (SEQ IDNO: 239) and 70 (SEQ ID NO: 240) CAGs on the normal and mutant Httallele, respectively. A SNP-based allele-specific real-time PCR assaywas first established to allow specific detection from the wild type orthe mutant Htt allele. The phasing of the SNP (r5363099 T/C) wasdetermined by Carroll, et al. (2011) Mol Ther. 19:2178-85; “T” is on thenormal allele and “C” is on the mutant allele. To detect Htt expressionfrom the mutant allele (099C), cDNA from the fibroblast was amplified byreal-time PCR (SsoFast EvaGreen Supermix, Bio-Rad) using forward primer099C.F (5′AGTTTGGAGGGTTTCTC, SEQ ID NO:143) and reverse primer 099.R5(5′ TCGACTAAAGCAGGATTTCAGG, SEQ ID NO:144); the annealing/extensiontemperature was 55.6° C. To detect Htt expression from the wild typeallele (099T), real-time PCR of the fibroblast cDNA were done usingforward primer 099T.F (5′AGTTTGGAGGGTTTCTT, SEQ ID NO:145), reverseprimer 099.R5 and the 3′phosphorylated blocker oligo 099T.BL(5′AGGGTTTCTCCGCTCAGC-3′phos, SEQ ID NO:146); the annealing/extensiontemperature was 58.3° C. Total human Huntingtin (hHtt, both wt andmutant allele) levels and normalization control beta-actin (ACTB) levelswere analyzed by real-time PCR using primer/probe Hs00918176_m1 and4352935E (Applied Biosystems), respectively. For the experiment shown inFIG. 3E, mRNA for indicated ZFPs were generated using the mMessagemMachine kit (Ambion), 1 ug of mRNA was transfected using Amaxanucleofector as above. Cells were harvested 48 hours after transfection;mRNA levels from the normal (CAG15 (SEQ ID NO: 239), 099T), the mutant(CAG70 (SEQ ID NO: 240), 099C) Htt allele and total Htt (hHtt) werequantified as described above and normalized to the levels of ACTB; theHtt/ACTB ratios for each sample was further normalized to that of themock-transfected sample. The “strong” CAG-targeted ZFP 30657 repressedboth alleles, as expected (based on its activity in HEK293 cells FIG.3A). ZFP 30640, which showed CAG repeat length-dependent repression ofthe reporters, gave <10% repression of the wild-type allele whilerepressed the mutant allele >90%. The levels of total Htt in each samplewere consistent with those of wt and mutant Htt levels in the samesample.

ZFP-30640 was also tested in a normal fibroblast line as well as otherHD fibroblast lines that contain different CAG repeat length in the Httgene (see FIG. 3F). Htt expression from each allele was detected asdescribed above. No Htt repression was observed in the normal fibroblastline (CAG18/18 (SEQ ID NOS 245 and 245)). In contrast, excellent allelicdiscrimination was observed in the CAG 15/67 (SEQ ID NOS 239 and 242)and CAG15/70 (SEQ ID NOS 239 and 240) lines at both a high and low doseof transfected 30640 mRNA; similar results were obtain for the two HDfibroblast lines with intermediate CAG repeat length on the mutantallele (CAG 18/44 (SEQ ID NOS 245 and 244) and CAG 18/45 (SEQ ID NOS 245and 243))—wherein the expanded allele is repressed by ˜80% at both thehigh and low doses of 30640, yet the CAG18 (SEQ ID NO: 245) alleleremained unaffected. Taken together, these data indicate thatallele-specific repressors such as 30640 can maintain strong CAG allelelength selectivity in the context of more prevalent disease genotypessuch as CAG18/44 (SEQ ID NOS 245 and 244) and CAG18/45 (SEQ ID NOS 245and 243).

Western blot analysis was used to show that that ZFPs such as 30640selectively down-regulated mutant Htt protein levels in twopatient-derived fibroblast lines, confirming allele-specific regulationthat was shown by qPCR assays (see FIG. 3G). ZFPs were delivered by mRNAtransfection (Amaxa nucleofection) at a 300 ng dose into 4 replicates of1.5e5 cells and pooled prior to plating in 12-well plates. At 48 hours,cells were washed and harvested for protein extract preparation.Approximately 2.5 ug of extract was loaded onto 5% Tris-acetate gels anddetected by MAB2166 (Millipore). Additionally, the same samples wereloaded on a 4-15% Tris-HCl gels (Bio-Rad) and transferred using standardmethods for detection by an anti B-Actin (1:20,000, Sigma) as loadingcontrols. Based on qPCR studies that measure Htt mRNA, 30640 is anallele-specific repressor targeting the CAG repeat; 32528 is biallelicrepressors targeting the transcription start site (TSS), and 30657 isCAG-targeted repressor that repress both Htt alleles at the dose thatwas used. Western blot showed that 30640 specifically reduced the levelsof mutant Htt (upper band) in both HD patient-derived cell lines, while32528 and 30657 repressed both alleles similarly.

Example 4: Additional CAG-Targeted ZFP Designs that Drive AlleleSpecific Repression of Htt

FIG. 4A shows the results of testing ZFP-TFs 30640, 30643, 30645 and33074 (all targeted to the CAG repeat and uses the KRAB repressiondomain) in a CAG18/45 (SEQ ID NOS 245 and 243) HD fibroblast line.Different amounts of ZFP mRNA were transfected using Amaxa nucleofectoras indicated, expression of the mutant Htt (right bar), wild type Htt(middle bar) and total Htt (both alleles, left bar) were measured asdescribed above at 24 hours after transfection. ZFPs 30640, 30645 and33074 drive allele-specific repression over the entire 3 ug-10 ng ZFPmRNA dose range; while 30643 appears to repress both allelessignificantly at doses that are 30 ng or higher, and begins to exhibitallele selectivity at the 10 ng dose.

FIG. 4B shows the results of testing ZFP 30643, 30648, 30657 and 30658(all targeted to CAG repeat and uses the KRAB repression domain) in aCAG15/70 (SEQ ID NOS 239 and 240) HD fibroblast line. Different amountsof ZFP mRNA were transfected using Amaxa nucleofector as indicated,expression of the mutant Htt (right bar), wild type Htt (middle bar) andtotal Htt (both alleles, left bar) were measured as described above at24 hours after transfection. Compared to the ZFPs that were tested inthe previous figure (30640, 30645 and 33074), these ZFPs drive mutantHtt-specific repression at lower doses. These results suggest thatdepend on ZFP expression levels that can be achieved in vivo (e.g. inthe brains of HD patients), allele-specific repression of mutant Htt canbe achieved using appropriate ZFP designs.

Example 5: Repression of Alternate CAG-Containing Genes

Using the RNAs isolated in Example 3 (FIG. 3E), repression of other CAGrepeat containing genes was analyzed, and the results are depicted inFIG. 5. The expression levels of the following genes was examined usingreal-time PCR and normalized to that of Actin: Ataxin 2 (“ATXN2”);Dynamin (“DNM1”); F-box only protein 11 (“FBXO11”), nitrate reductase(“NAP”); Origin recognition complex subunit 4 (“ORC4”); phosphokinase(“PHK”); OCT3/4 protein (“POU3”); THAP domain containing, apoptosisassociated protein 2 (“THAPII”); TATA binding protein (“TBP”); andstanniocalcin 1(“STC1”). In addition, the location of the CAG repeatsequence relative to the transcriptional start site (TSS) was noted, andis indicated in FIG. 3F as “TSS@”, where “+” indicates base position ofCAG repeats that are downstream of TSS, and “−” indicates base positionof CAG repeats that are upstream of TSS. Also, the number of CAG repeats(“#CAG”) is indicated for each gene.

The data demonstrate that repression of the mutant expanded Htt alleleby 30640 is highly specific, and only a subset of CAG repeat-containinggenes whose CAG repeats are relatively close to their respectivetranscroption start sites may be repression targets of ZFPs such as30640.

Example 6: Genome-Wide Specificity of Allele-Specific ZFP Repressors ofHtt

HD fibroblasts (CAG18/45 (SEQ ID NOS 245 and 243)) were transfected tostudy genome-wide specificity of CAG-targeted ZFPs by microarrayanalysis (FIG. 6). ZFPs were delivered by mRNA transfection (Amaxanucleofection) at the indicated doses—ZFPs 30640, 30645 and 33074 weretransfected in sextuplicate at the 75 ng, 15 ng and 15 ng dose,respectively; GFP-Kox mRNA (150 ng) was transfected as a control, andwas used as carrier to bring the total amount of transfected mRNA to 150ng in all samples. Expression from CAG18 (SEQ ID NO: 245) (099T, middlebars) and CAG45 (SEQ ID NO: 243) (099C, right bars) alleles was measuredby allele-specific qPCR reagents at 24 hours after transfection asdescribed above where each of the samples (1-6) are biologicalreplicates (separate transfections). Htt levels were normalized to thoseof GAPDH. Mutant allele-specific repression of Htt was observed for allthree ZFPs. The four most similar replicates were then chosen formicroarray analysis (Affymetrix HGU133plus2.0) as follows: GFP replicate1, 3, 4 and 6; 30640 replicate 2, 3, 5 and 6; 30645 replicate 2, 3, 5and 6; and 33074 replicate 1, 3, 4 and 5 were used for microarrayanalysis. Robust Multi-array Average (RMA) was used to normalize rawsignals from each probe set; ZFP-transfected samples were compared toGFP-transfected samples using T-test; “change” calls were made on genes(probe sets) with >2 fold difference relative to control samples andT-test P-value<0.05. Based on that criterion, 30640 repressed only twogenes, stanniocalcin 1 (STC1) and extended synaptotagmin-like protein 1(ESYT1); 30645 and 33074 only repressed one gene each, STC1 andinterleukin 17 receptor A (ILR17RA), respectively. Htt was not detectedas a repressed (>2-fold repression) gene because the Htt probe set onthe array detects both wt and mutant Htt mRNA. This experimentdemonstrates that mutant Htt-specific ZFPs, when expressed at levelsthat drive efficient allele-specific repression of Htt, can operate withvery high specificity genome-wide.

Example 7: Allele Specific Repression in HD Neural Stem Cells (NSCs)

HD iPSC/ESCs were passaged with accutase and cultured on matrigel coatedplates in E8 media (Life Technologies). Neural stem cells were derivedusing StemPro Neural Induction Medium (Life Technologies). Briefly,iPSC/ESCs were seeded into geltrex coated 6 well dish with 200,000cells/well and when 10-20% confluent the medium was changed to StemProNeural Induction Medium. Medium was changed every 2 days and NSCharvested and expanded on day 7. StemPro NSC SFM medium (LifeTechnologies) was used to culture NSCs. HD NSCs(CAG17/69 (SEQ ID NOS 236and 246), derived from Coriell GM23225 iPSC) were transfected with 1.5or 0.5 μg ZFP mRNA using nucleofection. Forty-eight hours posttransfection cells were harvested and expression quantified by RT-PCR.Allele-specific detection of Htt expression was performed using a SNP(rs1143646)-based genotyping assay #4351376 (Applied Biosystems). At theZFP doses that were tested, 30640 gave allele-specific repression ofmutant Htt, 30643 gave ˜50% repression of wt Htt and ˜90% repression ofmutant Htt, and 30648 repressed both alleles (FIG. 7); the behavior ofthese ZFPs is consistent with that in HD fibroblasts (FIG. 4). The totalHtt levels (middle bars) for each sample is consistent with levels ofmutant and wt Htt levels.

Example 8: Htt Repression in Differentiated HD Neurons

HD NSCs were passaged with accutase on geltrex coated plates. Neurondifferentiation was induced by changing medium to neural differentiationmedium containing StemPRO NSC SFM medium without (bFGF and EGF). Mediumwas changed every 3-4 days for up to 21 days. Neurons were derived fromNSC (CAG17/48 (SEQ ID NOS 236 and 247), derived from HD ESCs) by culturein neural differentiation medium. On day 15 post neural induction cellswere transfected with 1.0 or 0.5 μg ZFP mRNA using nucleofection.Forty-eight hours post transfection cells were harvested and geneexpression quantified by RT-PCR. Because this patient line does notcontain a SNP that allows qPCR-based allele-specific detection of wt andmutant Htt, only total Htt levels can be measured. Because we showedthat 30640 and 33074 does not repress the CAG18 (SEQ ID NO: 245) orCAG17 (SEQ ID NO: 236) allele in HD fibroblasts and NSCs, the levels oftotal Htt observed in 30640- and 33074-treated samples are consistentwith allele-specific repression of the mutant allele (CAG48 (SEQ ID NO:247)). More potent repression by 30643 and 30648 at the ZFP doses testedis also consistent with the behavior of these ZFPs in HD fibroblasts(FIG. 8).

Example 9: A CAG Targeted Repressor Represses Mutant Htt Transgene inR6/2 Mice

R6/2 mice (which carries a transgene of mutant human Htt exon 1 with 120CAG repeat (SEQ ID NO: 252), see Mangiarini, et al. (1996) Cell 15:197)received stereotactic, bilateral striatal injections of 3e10 vectorgenome of recombinant AAV2/6 encoding either ZFP 30640-KOX or GFP drivenby a CMV promoter. Mice were injected at 5 weeks of age and sacrificedfor molecular analysis at 8 weeks of age. Left and right striata weredissected from each hemisphere and snap frozen. To assess repression ofthe mutant Htt transgene, total RNA was extracted from each striatumwith TRIzol Plus (Life Technologies) followed by cDNA synthesis usingHigh Capacity RT (Life Technologies). Subsequently, R6/2 transgeneexpression was measured by qPCR and normalized to the geometric mean ofthree reference genes (Atp5b, Eif4a2, UbC) as previously described byBenn, et al. (2008) Molecular Neurodegeneration: 3:17. We observedstatistically significant repression (P<0.001) of the mutant Htttransgene in four ZFP-treated striata relative to the four GFP-treatedcontrol striata (FIG. 9). The average R6/2 repression was 64.9% of theGFP-treated controls. Because complete coverage of the striatum was notachieved using a single stereotactic injection and AAV2/6 preferentiallytransduces neuronal cells, the fold of repression observed (˜35%) islikely an underestimate of actual repression in cells that weretransduced with the AAV vector.

Example 10: Selective Repression of Mutant Htt Using ZFPs withDimerization/Multimerization Domains

In order to engineer zinc finger transcription factors to betterdiscriminate between short CAG and long GAG repeats, we sought to bothdecrease the DNA-binding affinity of individual zinc fingertranscription factors and increase the interaction strength betweendifferent copies of fusion protein bound to adjacent subsites within theCAG repeat. In order to decrease the DNA-binding affinity of individualzinc finger transcription factors, we generated zinc finger domains withfewer zinc fingers and/or with amino acid sequences expected to bind DNAwith less than optimal affinity. In order increase the interactionstrength between different copies of fusion protein bound to adjacentsubsites within the CAG repeat, we fused various dimerization domains toour zinc finger transcription factors. The dimerization domains caninteract in a “parallel” fashion and yield “head-to-head” or“tail-to-tail” dimers of fusion proteins that contain them. Onepotential dimerization strategy requires an array of identicalZFP-transcription factor fusions that bind in a “head-to-tail”orientation and thus this strategy requires dimerization domains thatinteract in an “anti-parallel” fashion. See, e.g., McClain, et al.(2001) J. Am. Chem. Soc. 123:3151-3152) and dimerizing zinc fingerpeptides (Giesecke, et al. (2006) Molecular Systems Biology2:2006-2011).

Dimerization contructs CC1 and CC2 were based on pairs of antiparallelcoiled coils (McClain, et al., ibid, Ghosh, et al. (2000) J Am Chem Soc122:5658-5659). Dimerization constructs CC3 and CC4 were truncatedversions of CC2 that lack either 4 residues or 7 residues respectively.Dimerization constructs DZ1, DZ2, DZ3, and DZ4 were based on pairs ofdimerizing zinc finger domains (Giesecke, et al., ibid). In each case,one member of the pair was fused to the N-terminus of the zinc fingerDNA binding domain and the other member of the pair was fused to theC-terminus of the zinc finger DNA binding domain. Short linkers rich inglycine and serine residues were used to fuse the dimerization domainsto the zinc finger binding domain. Additional embodiments of theinvention utilize linkers with alternate lengths and/or amino acidcomposition. Linkers with one or more residues removed or with one ormore glycine or serine residues changes to other amino acid residueswill reduce the flexibility of these linkers and may result in improveddiscrimination between long and short CAG repeats.

To achieve selective repression of the mutant Htt allele, ZFPs weredesigned as illustrated in FIG. 1D and FIGS. 10A and 4B. FIGS. 10C and10D show the sequences of the multimerization domains. FIGS. 11A and 11Bdepict the results that experiments designed to measure the ability ofthe ZFP-TFs comprising the CC and DZ domains, respectively to represstheir targets. For these experiments, indicated ZFP constructs (50 ng)were co-transfected with pRL-Htt-CAG17 (“CAG17” disclosed as SEQ ID NO:236) (200 ng), pGL3-Htt-CAG47 (“CAG47” disclosed as SEQ ID NO: 235) (200ng) and pVax-SEAP (secreted alkaline phosphatase, 10 ng, used asnormalization control) into HEK293 cells. Luciferase activity andsecreted alkaline phosphatase activities were measured 24 hours aftertransfection. The Renilla luciferase (CAG17 (SEQ ID NO: 236))/SEAP andfirefly luciferase (CAG47 (SEQ ID NO: 235))/SEAP ratios for each samplewere normalized to those of the reporter-only samples. ThepGL3-Htt-CAG47 (“CAG47” disclosed as SEQ ID NO: 235) reporter wasconstructed in the same way as the pRL-Htt-CAG47 (“CAG47” disclosed asSEQ ID NO: 235) reporter (see Example 3), except the pGL-promoterconstruct (Promega) was used instead of the pRL-TK construct.

As shown in FIG. 11A, 3 ZFPs, when tested asone or more CCdomain-containing constructs, enhanced repression of one or bothreporters when compared to constructs with the same ZFP but no CCdomains. FIG. 11B shows that DZ1 and DZ3 domains enhanced repression of32220 on both reporters.

These results suggest that the CC and DZ domains can in general increaseaffinity of multimerized ZFPs and that design of the DNA binding domainand the dimerization domain may yield optimal CAG-repeat lengthdiscrimination.

Example 11: Selective Repression of Mutant Htt with ZFP-ZFP-Kox Designs

ZFP TFs were tested in HD fibroblasts that were of the ZFP-ZFP-KOXdesign. In these experiments, the two ZFP DNA binding domains werelinked together with a flexible linker (LRQKDAARGSAAMAERPFQ, SEQ IDNO:179) and fused to a KOX repression domain. The linker was placedbetween the conserved histidines and cysteines. The proteins were testedas described above using ZFP mRNA at indicated doses. These results inthe CAG18/45 (SEQ ID NOS 245 and 243) (FIG. 12A) and CAG20/41 (SEQ IDNOS 248 and 249) (FIG. 12B) HD fibroblast lines demonstrated thatlinking less active ZFP DNA binding domains in this fashion can resultin composite ZFPs that drive allele-specific repression.

Example 12: Activation of Htt in Mouse Cells

ZFPs as described herein were also evaluated for their ability toactivate Htt expression. ZFPs targeted to the +200 to +467 bp region(relative to the transcription start site) of mouse Htt were fused tothe transcriptional activation domain of NFκB p65 subunit. Thistargeting region was chosen because this fragment was replaced by thecorresponding sequence (majority of exon 1 and some intron 1 sequence)from human Htt in various knock-in mouse models of HD (Menalled, et al.(2003) J. Comp. Neurol 4651:11-26; Wheeler, et al. (2000) Hum Mol Genet8:115-122), therefore ZFPs targeted to this region can selectivelyactivate the wild type allele in those animals but not the knock-inallele.

ZFPs were transfected into Neuro2A cells (both Htt alleles are wild typein these cells), mouse Htt and ACTB mRNA levels were measured asdescribed in Example 2 (duplicate transfections and multiple assays).

As shown in FIG. 13, an increase in Htt mRNA levels as compared to amock transfection was detected using both ZFP-TFs. See, FIG. 13A.Increased Htt protein levels were confirmed by Western blot. See, FIG.13B.

The generation of the knock-in Htt allele is illustrated in FIG. 13C;sequence alignment (FIG. 13D) shows divergence between the mousesequence that was replaced and the corresponding human sequence. FIG.13E shows that when such ZFP activators were transfected intoimmortalized striatal cells derived from HdhQ111/Q7 knock-in mice, onlythe wild type Htt was selectively activated.

Example 13: Regulation of Htt Expression In Vivo

To test efficacy of the Htt-specific ZFP TFs in vivo, AAV2 vectorsencoding the ZFPs are produced. These AAV2 based constructs are thendelivered to the brains of mice. For human Htt-specific ZFP TFs, AAVvectors are delivered to R6.2 mice or BAC HD mice (C57B1/6 or FVB/Nstrains) to assess the repression of the human transgene, as well aschange in HD-like phenotypes. For mouse Htt-specific ZFPs (activators orrepressors), AAV vectors are delivered to wild-type mice (C57B1/6 orFVB/N) or human Htt knock-in mice (HdhQ111/Q7, HdhQ140/Q7 or HdhQ175/Q7)to assess the activation or repression of the endogenous mouse Httexpression. For ZFPs that preferentially targeting the CAG-expandedallele, AAV vectors are delivered to R6.2 mice or human Htt knock-inmice (HdhQ111/Q7, HdhQ140/Q7 or HdhQ175/Q7) to examine the selectiverepression of wt vs. expanded Htt allele. Following sacrifice, braintissues are analyzed for Htt expression by Taqman real-time RT-PCR, anddemonstrate that the Htt genes are modulated by ZFP-TFs.

Example 14: Co-Transfection of a Neurotrophic Factor and a HD HttAllele-Specific ZFP TF

The Htt-specific ZFP TFs identified above are co-transfected with ZFPTFs-specific for a brain neurotrophic factor. The ZFP TF specific forbrain neurotrophic factors used are specific for either GDNF or BDNF.

Example 15: Design and Construction of Htt-Targeted Zinc FingerNucleases (ZFNs)

ZFNs targeting human Htt and mouse Htt were designed to target thesequences flanking the CAG repeats, sequences near the first coding ATG,the stop codon, as well as in early exons. ZFNs were designed andincorporated into plasmids or adenoviral vectors essentially asdescribed in Urnov, et al. (2005) Nature 435(7042):646-651; Perez, etal. (2008) Nature Biotechnology 26(7):808-816; and U.S. PatentPublication No. 2008/0131962.

Example 16: Cleavage Activity of Htt-Specific ZFNs

To test cleavage activity, plasmids encoding the pairs of humanHtt-specific ZFNs described above were transfected into K562 cells. K562cells were obtained from the American Type Culture Collection and grownas recommended in F-12 medium (Invitrogen) supplemented with 10%qualified fetal calf serum (FCS, Cyclone). Cells were disassociated fromplastic ware using TrypLE Select™ protease (Invitrogen). Fortransfection, one million K562 cells were mixed with 2 μg of thezinc-finger nuclease plasmid and 100 μL Amaxa Solution T. Cells weretransfected in an Amaxa Nucleofector II™ using program U-23 andrecovered into 1.4 mL warm F-12 medium+10% FCS.

Genomic DNA was harvested and a portion of the Htt locus encompassingthe intended cleavage site was PCR amplified. PCR using the AccuprimeHiFi polymerase from InVitrogen was performed as follows: after aninitial 3 minute denaturation at 94° C., 30 cycles of PCR are performedwith a 30 second denaturation step at 94° C. followed by a 30 secondannealing step at 58° C. followed by a 30 second extension step at 68°C. After the completion of 30 cycles, the reaction was incubated at 68°C. for 7 minutes, then at 10° C. indefinitely.

The genomic DNA from the K562 Htt-specific ZFN treated cells wasexamined by the Surveyor™ nuclease (Transgenomic) as described, forexample, in U.S. Patent Publication Nos. 2008/0015164; 2008/0131962; and2008/0159996.

Plasmids encoding the pairs of mouse Htt-specific ZFNs were tested insimilar fashion in Neuro-2a cells.

FIGS. 14A and B show that the ZFNs were capable of targeting the Httgenes with a gene modification efficiency of between 8-40%, assayed asdescribed previously by the amount of indels observed.

Example 17: Targeted Integration of Varying Lengths of TrinucleotideRepeats

The Htt-specific ZFNs with the greatest cleaving activity for sequencesflanking the CAG repeat as described above are used in a targetedintegration strategy to introduce varying lengths of CAG repeat into awild-type copy of Htt. Donors are constructed that contain 50 (SEQ IDNO: 230), 80 (SEQ ID NO: 231), 109 (SEQ ID NO: 232) and 180 (SEQ ID NO:233) repeat CAG units. These donors are then transfected into K562 cellswith plasmids encoding the Htt-specific ZFNs as described above.Verification of donor integration is achieved by genomic DNA isolation,PCR amplification (as described above) followed by sequencing of theregion of interest.

ZFNs identified in the K562 cells which result in targeted integrationof the donor alleles into the Htt allele are used to insert the variablelength donor nucleic acids into human embryonic stem cells (hESC).Successful donor integration is verified by genomic DNA isolation, PCRand sequencing as described above.

Example 18: Disruption/Knock-Out of Wild-Type and/or Mutant Htt

ZFNs that cleave in early exons can result in small insertion ordeletions (in-dels) as a result non-homologous end joining (NHEJ), thiscan generate cell models with one or both alleles of Htt disrupted,

Indicated ZFN pairs were prepared as described above and tested forcleavage activity using the Cel I mismatch as described for Example 8.These ZFN pairs target early exons of human Htt, and thus may be used toknock-out either the wild-type or a mutant Htt allele.

As shown in FIG. 14A, ZFP pairs 29627/29628, 29631/29632 (exon 12) and29637/29638 (exon 18) cleaved the Htt gene and can thus be utilized forgenerating knock-out cell lines.

Example 19: Expression Tagging of Wild-Type and HD Htt Alleles

ZFNs with the greatest cleaving activity for the first or last codingexon are used to tag the wild-type and mutant Htt allele with differentreporter proteins. Donor DNAs for each reporter (A and B) are designedbased on the cleavage site of the lead ZFN pair(s) to allow targetedintegration of the reporter gene to produce an in-frame fusion to Htt.Donor DNAs are co-transfected with the lead ZFN pair(s) into K562 cellsfor selecting the donor DNA construct that gives the highest frequencyof integration.

ZFN pairs were prepared as described above and tested for cleavageactivity using the Cel I mismatch as described for Example 8. The ZFNpairs used target the 3′ end of the Htt coding sequence, and thus may beused to target either a wild-type or a mutant Htt allele. As shown inFIG. 14B, ZFP pairs 25917/25916, 25920/25921 and 25923/25922 werecapable of cleaving the Htt gene and can thus be utilized for theintroduction of a reporter tag.

The selected donor DNA construct for reporter A along with correspondingZFNs are delivered to cells derived from subjects carrying mutant Httgene (e.g. fibroblasts, induced pluripotent cells) Clones are derivedand screened for the target integration of the reporter A. Heterozygousevents are desired and the targeted alleles are identified by PCR.Clones containing a single reporter-tagged Htt allele and unmodified ZFNtarget sequence on the other allele are selected; the donor constructfor reporter B and corresponding ZFNs are transfected to tag the secondallele with the reporter B.

The resulting mouse embryonic stem cell clone contains the wild-type Httallele and mutant allele tagged with two different markers that allowtracking of expression from each allele; these cells are used togenerate mouse models of trinucleotide repeat disorders using standardprotocols.

Example 20: Construction of Active TALE-TF Proteins Against Htt

TALE DNA binding domains were linked to the KRAB repression domain fromthe Kox1 protein (TALE TF) and used to test repression of the Htt genein HD patient (CAG 20/41 (SEQ ID NOS 248 and 249))-derived fibroblasts.The construction of the TALE proteins was done as described previously(see co-owned U.S. Patent Publication No. 2011/0301073 and co-owned U.S.Pat. No. 9,458,205, both of which are incorporated herein by reference),and were constructed with three different C-terminal architectures: +63,+231 and +278 as described in U.S. Patent Publication No. 2011/0301073.To construct the TALE TF expression plasmids, the TALEN expressionplasmids described previously (see U.S. Patent Publication No.2011/0301073) were used except that the Fok I domain used for in theTALENs was replaced with the KRAB repression domain. The linkages of theC-terminus of the TALE protein and the KRAB domain are shown below,where the KRAB domain sequence is indicated by underline. The bold anditalicized text indicates the triple flag tag, the bold text indicatesthe nuclear localization sequence, “[repeats]” indicates the location ofthe TALE repeat unit array (full repeats plus the C-terminal halfrepeat), and the wavy underlined portion shows the sequence of the KRABdomain:

TALE - C63 - Kox1: (SEQ ID NOS 197 & 210) M

MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEI KSSVTALE - C231 - Kox1: (SEQ ID NOS 198 & 211) M

MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV TALE - C278 - Kox1 (SEQ ID NOS 199 & 212) M

MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREI HQETHPDSETAFEIKSSV

Base recognition was achieved using the canonical RVD-basecorrespondences (the “TALE code”: NI for A, HD for C, NN for G (NK inhalf repeat), NG for T). In some of the TALE TFs, the protein isdesigned to bind the sense (5′-3′) strand of the DNA, while in others,the TALE TF is designed to bind to the anti-sense (3′-5′) strand. Thisset of TALE TFs was designed to target the CAG repeats of the Htt gene.TALE DNA binding proteins often preferentially interact with a ‘T’nucleotide base at the 5′ end of the target, and so since the targetsare CAG repeat regions, it can be predicted that the proteins that bindto the anti-sense DNA strand, and thus CTG repeat sequences with thebase ‘T’ at the 5′ 3nd of the target, might have better binding affinityand specificity and thus repressor activity.

The targets and numeric identifiers for the TALE TFs tested are shownbelow in Table 4. Numeric identifiers are labeled “SBS#”, specificityfor the Sense or Antisense strand is indicated (“S/A”), as well as thetarget, the number of repeat units or RVDs and the type of C-terminus.

TABLE 4 Htt specific TALE-TFs SEQ ID SBS# S/A Target (5′ - 3′) NO RVDsC term 102449 S gcAGCAGCAGCAGCAGCAGca 200 17 +63 102450 SgcAGCAGCAGCAGCAGca 201 14 +63 102451 S gcAGCAGCAGCAGca 202 11 +63 102452S gcAGCAGCAGca 203 8 +63 102453 A ctGCTGCTGCTGCTGCTGCtg 204 17 +63102454 A ctGCTGCTGCTGCTGCtg 205 14 +63 102455 A ctGCTGCTGCTGCtg 206 11+63 102456 A ctGCTGCTGCtg 207 8 +63 102457 S gcAGCAGCAGCAGCAGCAGca 20017 +231 102458 S gcAGCAGCAGCAGCAGca 201 14 +231 102459 S gcAGCAGCAGCAGca202 11 +231 102460 S gcAGCAGCAGca 203 8 +231 102462 A ctGCTGCTGCTGCTGCtg205 14 +231 102463 A ctGCTGCTGCTGCtg 206 11 +231 102464 A ctGCTGCTGCtg207 8 +231 102466 S gcAGCAGCAGCAGCAGca 201 14 +278 102467 SgcAGCAGCAGCAGca 202 11 +278 102468 S gcAGCAGCAGca 203 8 +278 102469 ActGCTGCTGCTGCTGCTGCtg 204 17 +278 102470 A ctGCTGCTGCTGCTGCtg 205 14+278 102471 A ctGCTGCTGCTGCtg 206 11 +278 102472 A ctGCTGCTGCtg 207 8+278

The TALE TFs in the table were then tested for Htt repression in HDpatient (CAG 20/41 (SEQ ID NOS 248 and 249)) derived fibroblasts, andthe results are shown in FIG. 15. In this experiment, the cells weretransfected with either 1000, 100 or 10 ng of TALE-TF encoding mRNA. Theresults for each TALE TF assayed are shown in groups of three,representing the three transfected mRNA amounts. In each grouping, thereare also three samples: the left bar indicates the total Htt expression,the middle bar indicates the expression from the CAG20 (SEQ ID NO: 248)Htt allele, and the right bar indicates the expression from the CAG41(SEQ ID NO: 249) Htt allele. The data demonstrates that there are someTALE TFs that were able to repress both Htt alleles (see for example102454), while other TALE TFs were able to selectively inhibit themutant Htt with the extended CAG repeat (see for example 102451 and102472).

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A non-naturally occurring zinc finger proteincomprising 6 zinc finger domains ordered F1 to F6, respectively, whereinthe zinc finger protein specifically binds to mutant Huntington's geneallele (mHtt) gene and comprises the recognition helix regions asfollows: (SEQ ID NO: 60) F1: RSDNLSE; (SEQ ID NO: 61) F2: KRCNLRC;(SEQ ID NO: 18) F3: QSGDLTR; (SEQ ID NO: 18) F4: QSGDLTR;(SEQ ID NO: 60) F5: RSDNLSE; and (SEQ ID NO: 61) F6: KRCNLRC.


2. The zinc finger protein of claim 1, wherein the zinc finger proteinbinds to sequences within the CAG repeat region of the mHtt gene.
 3. Afusion protein comprising a zinc finger protein of claim 1 and atranscriptional repression domain.
 4. A polynucleotide encoding one ormore fusion proteins according to claim
 3. 5. An AAV vector comprisingthe polynucleotide of claim
 4. 6. The AAV vector of claim 5, wherein theAAV vector is an AAV6 or AAV9 vector.
 7. A host cell comprising one ormore zinc finger proteins of claim
 1. 8. A pharmaceutical compositioncomprising one or more zinc finger proteins according to claim
 1. 9. Apharmaceutical composition comprising one or more polynucleotidesaccording to claim
 4. 10. A pharmaceutical composition comprising one ormore AAV vectors according to claim
 5. 11. A method of modifyingexpression of an mHtt gene in a cell, the method comprisingadministering to the cell one or more polynucleotides encoding one ormore fusion proteins according to claim
 3. 12. A method of treatingHuntington's Disease, the method comprising administering one or morepolynucleotides according to claim 4 to a subject in need thereof.
 13. Amethod of treating Huntington's Disease, the method comprisingadministering one or more AAV vectors according to claim 5 to a subjectin need thereof.